Keto-isovalerate decarboxylase enzymes and methods of use thereof

ABSTRACT

Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 61/512,866, filed Jul. 28, 2011, herein incorporated by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Agreement DE-AR0000006 awarded by the United States Department of Energy. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to polypeptides having α-keto-isovalerate decarboxylase activity suited for performance in isobutanol production pathways.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

Methods for the chemical synthesis of the butanol isomer isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A. Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals. The production of isobutanol from plant-derived raw materials could minimize the use of fossil fuels and would represent an advance in the art.

U.S. Pat. No. 7,851,188 describes enzymatic pathways for the production of isobutanol in recombinant microorganisms. The penultimate step in one metabolic pathway described therein for the production of isobutanol is the conversion of α-ketoisovalerate to isobutyraldehyde. There remains a need in the art to identify additional enzymes that are suitable for use in isobutanol biosynthetic pathways.

SUMMARY OF THE INVENTION

Provided herein are recombinant host cells and methods of converting α-ketoisovalerate to isobutyraldehyde employing the polypeptides disclosed. In embodiments, methods comprise: (a) providing a polypeptide wherein said polypeptide comprises at least one of: (i) at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof; or (ii) α-ketoisovalerate decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than about 1 and thiamine diphosphate cofactor activation constant (K_(c)) of about 20 μM or less; and (b) contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63. In embodiments, the polypeptide comprises a sequence from Listeria grayi or Macrococcus caseolyticus. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 58 or 61. In embodiments, the contacting occurs in the presence of less than about 30 mg/L, less than about 20 mg/L, or less than about 10 mg/L thiamine. In embodiments, the contacting occurs within a recombinant host cell and wherein the polypeptide is heterologous to recombinant host cell. In embodiments, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In embodiments, the recombinant host cell is Saccharomyces cerevisiae. In embodiments, the recombinant host cell further comprises heterologous polynucleotides encoding polypeptides which catalyze the substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; and (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In embodiments, the host cell further comprises a heterologous polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol. In embodiments, the recombinant host cell further comprises reduced or eliminated pyruvate decarboxylase activity. In embodiments, the recombinant host cell further comprises at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis. In embodiments, the recombinant host cell comprises deletion of fra2. In embodiments, the recombinant host cell comprises reduced or eliminated glycerol-3-phosphate dehydrogenase activity.

Provided herein also are methods of producing isobutanol comprising: (a) providing a recombinant host cell comprising an isobutanol production pathway, the production pathway comprising a polypeptide wherein said polypeptide comprises at least one of: (i) at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof; or (ii) α-ketoisovalerate decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than 1, and thiamine diphosphate cofactor activation constant (K_(c)) of about 20 μM or less; and (b) contacting the recombinant host cell with a carbon substrate under conditions whereby isobutanol is produced. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63. In embodiments, the polypeptide comprises a sequence from Listeria grayi or Macrococcus caseolyticus. In embodiments, the polypeptide has a KIVD cluster profile HMM E value of less than 1E-223 using the hmmsearch program. In embodiments, the methods further comprise isolating the isobutanol, and in embodiments, isolating comprises liquid-liquid extraction. In embodiments, the extractant for liquid-liquid extraction comprises C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, and mixtures thereof. In embodiments, the recombinant host cell is Saccharomyces cerevisiae. In embodiments, methods of producing isobutanol provided herein comprise: (a) providing a recombinant host cell comprising an isobutanol biosynthetic comprising a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 52 or 61; and (b) contacting the recombinant host cell with a carbon substrate under conditions whereby isobutanol is produced. In embodiments, the contacting occurs in the presence of less than about 30 g/L thiamine.

In embodiments, methods and recombinant host cells provided herein comprise polypeptides having at least about 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, 63, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, or 416, or an active fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS AND INCORPORATION OF THE SEQUENCE LISTING AND TABLE FILED ELECTRONICALLY HEREWITH

FIG. 1 depicts specificity ratio data for the substrates α-ketoisovalerate and pyruvate for example KIVD polypeptides (from left to right: Mca, L. lactis, KdcA, kivD81).

FIG. 2 demonstrates yields of α-ketoisovalerate and pyruvate in the presence of 0 mg/L, 1 mg/L and 30 mg/L thiamine for recombinant host cells as described in the Examples.

FIG. 3 shows the concentration of α-ketoisovalerate over the fermentation time for recombinant host cells in the presence of 0, 1 mg/L, and 30 mg/L thiamine as described in the Examples.

Table Z filed electronically herewith and incorporated by reference herein in its entirety is the KIVD cluster Profile HMM described herein. Table Z forms part of the specification.

The sequences provided in the sequence listing filed herewith (20120727_CL5253USNP_SEQ_ST25.txt; Size: 1,304,771 bytes; Creation date: Jul. 26, 2012), herein incorporated by reference, conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with the World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(α-βis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

In order to further define this invention, the following terms and definitions are herein provided.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers may be added to the specified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. §2111.03.

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “isobutanol biosynthetic pathway” refers to the enzymatic pathway to produce isobutanol. From time to time “isobutanol biosynthetic pathway” is used synonymously with “isobutanol production pathway”.

The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by the recombinant host cells disclosed herein. Non-limiting examples of carbon substrates are provided herein and include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, glucose, fructose, sucrose, xylose, arabinose, dextrose, one-carbon substrates or mixtures thereof.

The term “effective titer” as used herein, refers to the total amount of isobutanol produced by fermentation per liter of fermentation medium. The total amount of isobutanol includes: (i) the amount of isobutanol in the fermentation medium; (ii) the amount of isobutanol recovered from the fermentation medium, for example, by contact with an organic extractant; and (iii) the amount of isobutanol recovered from the gas phase, if gas stripping is used.

The term “effective rate” as used herein, refers to the total amount of isobutanol produced by fermentation per liter of fermentation medium per hour of fermentation.

The term “effective yield” as used herein, refers to the amount of isobutanol produced per unit of fermentable carbon substrate consumed by the biocatalyst.

The term “acetolactate synthase” refers to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO₂. Acetolactate has two stereoisomers ((R) and (S)); the enzyme prefers the (S)-isomer, which is made by biological systems. Acetolactate synthases may be classified as EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego).

The term “ketol-acid reductoisomerase” (abbreviated “KARI”), and “acetohydroxy acid isomeroreductase” will be used interchangeably and refer to enzymes capable of catalyzing the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego).

The terms “acetohydroxy acid dehydratase” and “dihydroxyacid dehydratase” (DHAD) refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Acetohydroxy acid dehydratases may be classified as EC number 4.2.1.9 and are available from a vast array of microorganisms.

The term “branched-chain α-keto acid decarboxylase” or “α-ketoacid decarboxylase” or “α-ketoisovalerate decarboxylase” or “2-ketoisovalerate decarboxylase” (herein also referred to as ketoisovalerate decarboxylase or, from time to time, KIVD) refers to an enzyme that catalyzes the conversion of α-ketoisovalerate (“α-Kiv”) to isobutyraldehyde and CO₂. Ketoisovalerate decarboxylase sequences are available from a number of microorganism sources, including those disclosed herein.

The term “branched-chain alcohol dehydrogenase” refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Branched-chain alcohol dehydrogenases may be classified as EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2).

The term “KIVD cluster Profile HMM” refers to the Profile Hidden Markov Model prepared as disclosed herein. The KIVD cluster Profile HMM is provided as Table Z.

The term “isolated nucleic acid molecule”, “isolated nucleic acid fragment” and “genetic construct” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:

TABLE 1 Amino Acids Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Leucine Leu L Isoleucine Ile I Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of a microorganism. A “foreign” gene refers to a gene not normally found in the host microorganism, but that is introduced into the host microorganism by gene transfer. For example, foreign genes can comprise native genes inserted into a non-native microorganism, or chimeric genes. Foreign genes can also comprise, for example, native genes with mutations that change an amino acid residue of an encoded polypeptide. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

As used herein the term “coding sequence” refers to a DNA sequence that encodes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term “endogenous,” when used in reference to a polynucleotide, a gene, or a polypeptide refers to a native polynucleotide or gene in its natural location in the genome of an organism, or for a native polypeptide, is transcribed and translated from this location in the genome.

The term “heterologous” when used in reference to a polynucleotide, a gene, or a polypeptide refers to a polynucleotide, gene, or polypeptide not normally found in the host organism. “Heterologous” also includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. The heterologous polynucleotide or gene may be introduced into the host organism by, e.g., gene transfer. A heterologous gene may include a native coding region with non-native regulatory regions that is reintroduced into the native host. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

The term “recombinant genetic expression element” refers to a nucleic acid fragment that expresses one or more specific proteins, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ termination sequences) coding sequences for the proteins. A chimeric gene is a recombinant genetic expression element. The coding regions of an operon may form a recombinant genetic expression element, along with an operably linked promoter and termination region.

“Regulatory sequences” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, operators, repressors, transcription termination signals, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term “promoter” refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. “Inducible promoters,” on the other hand, cause a gene to be expressed when the promoter is induced or turned on by a promoter-specific signal or molecule. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. For example, it will be understood that “FBA1 promoter” can be used to refer to a fragment derived from the promoter region of the FBA1 gene.

The term “terminator” as used herein refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence. It is recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical terminator activity. For example, it will be understood that “CYC1 terminator” can be used to refer to a fragment derived from the terminator region of the CYC1 gene.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host microorganism, resulting in genetically stable inheritance. Host microorganisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” microorganisms.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine, and with respect to RNA, adenine is complementary to uracil and cytosine is complementary to guanine.

Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 2. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

TABLE 2 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L) TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S) TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available in the art, for example, at the “Codon Usage Database” available at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 3. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. Table 1B has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

TABLE 3 Codon Usage Table for Saccharomyces cerevisiae Frequency per Amino Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 120510 18.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC 35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 Ile AUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 144243 22.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU 153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 Ser AGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8 Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 83207 12.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC 82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 Tyr UAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3 Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA 273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2 Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 31095 4.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 19562 3.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU 156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 Stop UAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art. Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as “synthetic gene designer” (userpages.umbc.edu/˜wug1/codon/sgd/, visited Mar. 19, 2012).

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as Basic Local Alignment Search Tool (BLAST) (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data. Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, such as from other species, wherein such polypeptides have the same or similar function or activity, or in describing the corresponding polynucleotides. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable polynucleotide fragments not only have the above homologies but typically comprise a polynucleotide having at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, or at least 250 nucleotides. Further, suitable polynucleotide fragments having the above homologies encode a polypeptide having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.](1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Other molecular tools and techniques are known in the art and include splicing by overlapping extension polymerase chain reaction (PCR) (Yu, et al. (2004) Fungal Genet. Biol. 41:973-981), positive selection for mutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J. D. et al. (1984) Mol. Gen. Genet. 197, 345-346; M A Romanos, et al. Nucleic Acids Res. 1991 Jan. 11; 19(1): 187), the cre-lox site-specific recombination system as well as mutant lox sites and FLP substrate mutations (Sauer, B. (1987) Mol Cell Biol 7: 2087-2096; Senecoff, et al. (1988) Journal of Molecular Biology, Volume 201, Issue 2, Pages 405-421; Albert, et al. (1995) The Plant Journal. Volume 7, Issue 4, pages 649-659), “seamless” gene deletion (Akada, et al. (2006) Yeast; 23(5):399-405), and gap repair methodology (Ma et al., Genetics 58:201-216; 1981).

As disclosed herein, Applicants have discovered polypeptides not previously annotated as α-ketoisovalerate decarboxylases polypeptides capable of catalyzing the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde. Polynucleotides encoding the putative decarboxylases were synthesized, expressed in E. coli, and tested for α-ketoisovalerate decarboxylase activity. As shown in the examples. certain polypeptides were employed in recombinant host cells comprising an isobutanol biosynthetic pathway.

KIVD Polypeptides

Members of the protein family of ketoisovalerate decarboxylase (KIVD) were identified through National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov, visited Jul. 27, 2012) BLAST searches of NCBI non-redundant (nr) protein database using amino acid sequences of L. lactis KivD (alpha-ketoisovalerate decarboxylase) (SEQ ID NO: 68), L. lactis KDCA (branched-chain keto acid decarboxylase) (SEQ ID NO: 66), Enterobacter cloacae IPDC (Indolepyruvate decarboxylase) (SEQ ID NO: 257) with the following search parameters: E value=10, word size=3, Matrix=Blosum62, and Gap opening=11 and gap extension=1, E value cutoff of 10⁻³. The three blast result sets were combined and sequences that are identical or whose lengths are greater than 712 or less than 583 were removed. This combined sequence set was then reduced to a set where the maximum identity between sequences is 65% (“nr65 set”) using the program CD-Hit (downloaded January, 2007; available at weizhong-lab.ucsd.edu/cd-hit/, visited Jul. 25, 2012). The resulted in a set of 1184 KIVD sequences.

The 1184 sequences were aligned using ClustalW using all default parameters. A phylogenetic tree was then constructed from the multiple sequence alignment with neighbor-joining program using ClustalW. The phylogenetic tree was visualized using program iTOL (itol.embl.de, visited Jul. 27, 2012). A subtree of 180 sequences was selected which spanned over L. Lactis KivD, E. cloacae IPDC, S. cerevisiae PDC (pyruvate decarboxylase) and Zymomonas PDC and their close homologs. This subset was then expanded to include all cluster members in the nr65 set of these 180 sequences. The final set consisted of 601 sequences and was used to construct the sequence similarity network as described below.

The sequence similarity network consisted of a collection of edges corresponding to pairwise relationships that are better than a defined threshold (Atkinson H J et al. PLoS One. 2009, 4:e4345). For the analysis here, pairwise relationships correspond to BLAST alignments associated with an E-value. The set of 601 sequences were used to create a custom BLAST database using ‘formatdb’ (NCBI; www.ncbi.nlm.nih.gov/BLAST/docs/formatdb.html; visited Jul. 26, 2012). Then each sequence in the set was searched against this database using BLAST. Since BLAST E-values are not symmetric, the best E-value was kept and associated with each pairwise comparison.

All pairwise alignments that were better than E-value threshold of E-130 were loaded into program Cytoscape (www.cytoscape.org; visited Jul. 27, 2012), and a graph was generated using the Organic layout algorithm. Upon visualization, these 601 sequences fell into discernable clusters. One cluster containing 170 sequences was identified to contain L. lactis KivD, L. lactis KDCA, and E. cloacae IPDC. Since L. lactis KivD and L. lactis KDCA are known α-ketoisovalerate decarboxylases, several candidates from this cluster were selected for the diversity selection. Certain sequences from this cluster were experimentally verified as shown herein to have α-ketoisovalerate decarboxylase activity (See Examples 3 and 5).

Because the cluster was verified to contain polypeptides with □-ketoisovalerate decarboxylase activity, a profile HMM for the KIVD cluster was generated from the set of 170 sequences described above and provided in Table 4. The % identity, as determined from the multiple sequence alignment by ClustalW, of the cluster sequences to sequences verified experimentally to have □-etoisovalerate decarboxylase activity is provided in Table 4. One of skill in the art will appreciate that polypeptides provided in Table 4 are candidate polypeptides for embodiments provided herein and can readily make and test the polypeptides for □-ketoacid decarboxylase activity. Accordingly source organisms provided in Table 4 may be source organisms for polypeptides suitable for embodiments provided herein. Thus, provided herein are methods for converting α-ketoisovalerate to isobutyraldehyde comprising providing a polypeptide comprising SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, 63, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, or 416 (or a polypeptide having at least 80%, 85%, 90%, 95%, or 98% identity thereto or an active fragment thereof) and contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced. In some embodiments, said contacting occurs within a recombinant host cell. In some embodiments, said host cell comprises an isobutanol biosynthetic pathway. In some embodiments, recombinant host cells comprise a polynucleotide encoding a polypeptide of Table 4, or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto, or an active fragment thereof. Provided herein are methods for producing isobutanol comprising providing a recombinant host cell comprising a polypeptide comprising SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, 63, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, or 416 (or a polypeptide having at least 80%, 85%, 90%, 95%, or 98% identity thereto or an active fragment thereof) and contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced.

TABLE 4 Percent Identity of KIVD cluster sequences Identity to SEQ ID NO: (GI number) Cluster 51 52 53 55 56 58 59 61 63 KIVD cluster member Source organism Member SEQ ID NO: (240171442) (229556973) (228908218) (118464281) (71065418) (294497944) (291276462) (222151578) (16417060) gi|228943180| Bacillus 328 36% 43% 93% 40% 38% 62% 50% 44% 40% ref|ZP_04105648.1 thuringiensis serovar berliner ATCC 10792 gi|229145075| Bacillus 342 37% 44% 94% 40% 39% 62% 50% 44% 41% ref|ZP_04273468.1 cereus BDRD- ST24 gi|229044211| Bacillus 333 37% 43% 94% 40% 39% 62% 50% 44% 41% ref|ZP_04191886.1 cereus AH676 gi|229069982| Bacillus 334 36% 44% 91% 40% 38% 63% 49% 44% 41% ref|ZP_04203259.1 cereus F65185 gi|229079646| Bacillus 335 36% 43% 92% 40% 38% 63% 49% 44% 41% ref|ZP_04212180.1 cereus Rock4-2 gi|206971696| Bacillus 306 36% 44% 92% 40% 38% 63% 49% 44% 41% ref|ZP_03232646.1 cereus AH1134 gi|228952828| Bacillus 330 36% 43% 92% 40% 38% 62% 49% 43% 41% ref|ZP_04114898.1 thuringiensis serovar kurstaki str. T03a001 gi|229181920| Bacillus 346 36% 43% 93% 40% 38% 63% 49% 44% 41% ref|ZP_04309225.1 cereus 172560W gi|229109914| Bacillus 338 37% 43% 92% 40% 38% 62% 50% 44% 41% ref|ZP_04239496.1 cereus Rock1-15 gi|218230913| Bacillus 312 36% 43% 91% 40% 39% 63% 49% 44% 41% ref|YP_002367156. cereus B4264 gi|30020564| Bacillus 398 37% 44% 92% 40% 39% 62% 50% 44% 41% ref|NP_832195.1| cereus ATCC 14579 gi|229127869| Bacillus 341 37% 44% 92% 40% 39% 62% 49% 44% 41% ref|ZP_04256855.1 cereus BDRD-Cer4 gi|229150678| Bacillus 343 37% 43% 94% 40% 39% 62% 50% 44% 41% ref|ZP_04278892.1 cereus m1550 gi|229190623| Bacillus 347 36% 44% 93% 40% 39% 63% 50% 44% 40% ref|ZP_04317620.1 cereus ATCC 10876 gi|228908218| Bacillus 53 37% 44% 100% 40% 39% 63% 50% 44% 41% ref|ZP_04072064.1 thuringiensis IBL 200 gi|30262484| Bacillus 399 37% 44% 90% 40% 38% 62% 50% 44% 42% ref|NP_844861.1| anthracis str. Ames gi|170704700| Bacillus 293 37% 44% 89% 40% 38% 63% 50% 44% 42% ref|ZP_02895166.1 anthracis str. A0389 gi|165868862| Bacillus 283 37% 44% 89% 40% 38% 63% 51% 44% 42% |ref|ZP_02213522.1 anthracis str. A0488 gi|228946089| Bacillus 329 38% 44% 90% 41% 38% 63% 50% 45% 42% ref|ZP_04108425.1 thuringiensis serovar monterrey BGSC 4AJ1 gi|49477724| Bacillus 404 38% 45% 91% 41% 39% 63% 51% 45% 42% ref|YP_036605.1| thuringiensis serovar konkukian str. 97-27 gi|228933783| Bacillus 327 38% 44% 90% 41% 39% 63% 51% 45% 42% ref|ZP_04096629.1 thuringiensis serovar andalousiensis BGSC 4AW1 gi|196033775| Bacillus 300 38% 45% 90% 41% 39% 64% 51% 45% 42% ref|ZP_03101186.1 cereus W gi|218903623| Bacillus 313 38% 45% 90% 41% 39% 64% 51% 45% 42% ref|YP_002451457. cereus AH820 gi|229122031| Bacillus 340 38% 44% 91% 41% 39% 63% 51% 45% 42% ref|ZP_04251247.1 cereus 95/8201 gi|52143006| Bacillus 408 38% 45% 90% 41% 39% 63% 51% 45% 42% ref|YP_083823.1| cereus E33L gi|228915074| Bacillus 326 38% 44% 90% 41% 38% 63% 51% 45% 42% ref|ZP_04078671.1 thuringiensis serovar pulsiensis BGSC 4CC1 gi|229196690| Bacillus 348 38% 44% 91% 41% 39% 63% 51% 45% 42% ref|ZP_04323433.1 cereus m1293 gi|217959977| Bacillus 311 38% 44% 89% 41% 39% 63% 51% 46% 42% ref|YP_002338533. cereus AH187 gi|206973990| Bacillus 307 37% 44% 89% 41% 39% 63% 51% 45% 42% ref|ZP_03234908.1 cereus H3081.97 gi|222096064| Bacillus 315 38% 44% 90% 41% 39% 63% 51% 45% 42% ref|YP_002530121. cereus Q1 gi|228985570| Bacillus 331 37% 44% 90% 40% 39% 62% 51% 45% 42% ref|ZP_04145724.1 thuringiensis serovar tochigiensis BGSC 4Y1 gi|229156064| Bacillus 344 37% 43% 89% 40% 39% 61% 50% 45% 42% ref|ZP_04284163.1 cereus ATCC 4342 gi|47570048| Bacillus 403 37% 43% 90% 40% 39% 63% 50% 45% 41% ref|ZP_00240709.1| cereus G9241 gi|229173153| Bacillus 345 37% 44% 91% 41% 39% 62% 50% 44% 42% ref|ZP_04300703.1 cereus MM3 gi|229030189| Bacillus 332 38% 44% 90% 41% 39% 63% 50% 44% 42% ref|ZP_04186249.1 cereus AH1271 gi|42781579| Bacillus 401 37% 44% 90% 41% 39% 62% 51% 44% 42% ref|NP_978826.1| cereus ATCC 10987 gi|229100258| Bacillus 336 37% 44% 86% 40% 40% 63% 50% 46% 42% ref|ZP_04231149.1 cereus Rock3- 29 gi|229115945| Bacillus 339 37% 44% 87% 41% 40% 63% 50% 46% 42% ref|ZP_04245341.1 cereus Rock1-3 gi|229103080| Bacillus 337 38% 44% 88% 41% 40% 63% 50% 45% 42% ref|ZP_04233768.1 cereus Rock3- 28 gi|294497944| Bacillus 58 40% 44% 63% 42% 41% 100% 50% 48% 44% ref|YP_003561644. megaterium QM B1551 gi|15004729| Clostridium 266 40% 44% 61% 41% 41% 69% 48% 44% 43% ref|NP_149189.1| acetobutylicum ATCC 824 gi|186682481| Nostoc 297 37% 39% 48% 38% 35% 48% 44% 40% 35% ref|YP_001865677. punctiforme PCC 73102 gi|16417060| Sarcina 63 34% 38% 41% 35% 35% 44% 48% 41% 100% gb|AAL18557.1| ventriculi AF35 gi|291276462| Helicobacter 59 39% 40% 50% 41% 39% 50% 100% 44% 48% ref|YP_003516234. mustelae 12198 gi|27469128| Staphylococcus 381 35% 44% 44% 36% 38% 46% 43% 66% 40% ref|NP_765765.1| epidermidis ATCC 12228 gi|251811671| Staphylococcus 361 35% 44% 44% 36% 38% 46% 43% 66% 40% ref|ZP_04826144.1 epidermidis BCM-HMP0060 gi|242243554| Staphylococcus 359 34% 44% 44% 36% 38% 47% 42% 65% 39% ref|ZP_04797999.1 epidermidis W23144 gi|242372336| Staphylococcus 360 34% 43% 43% 35% 38% 46% 41% 65% 39% ref|ZP_04817910.1 epidermidis M23864:W1 gi|223044198| Staphylococcus 316 34% 44% 44% 35% 38% 47% 41% 64% 40% ref|ZP_03614236.1 capitis SK14 gi|239637821| Staphylococcus 358 34% 43% 43% 35% 39% 46% 41% 67% 39% ref|ZP_04678783.1 warneri L37603 gi|289549575| Staphylococcus 389 35% 46% 45% 37% 39% 46% 42% 65% 39% ref|YP_003470479. lugdunensis HKU09-01 gi|253734750| Staphylococcus 364 34% 44% 44% 35% 39% 45% 41% 65% 39% ref|ZP_04868915.1 aureus subsp. aureus TCH130 gi|21281891| Staphylococcus 308 34% 44% 44% 35% 39% 45% 41% 65% 39% ref|NP_644977.1| aureus subsp. aureus MW2 gi|283469432| Staphylococcus 384 34% 45% 44% 35% 39% 45% 41% 65% 39% emb|CAQ48643.1| aureus subsp. aureus ST398 gi|253730541| Staphylococcus 363 34% 44% 44% 35% 39% 45% 41% 65% 40% ref|ZP_04864706.1 aureus subsp. aureus USA300_TCH959 gi|57651189| Staphylococcus 410 34% 44% 43% 35% 39% 45% 41% 66% 40% ref|YP_185072.1| aureus subsp. aureus COL gi|15923178| Staphylococcus 280 34% 44% 43% 36% 38% 45% 41% 65% 40% ref|NP_370712.1| aureus subsp. aureus Mu50 gi|148266613| Staphylococcus 264 34% 44% 43% 36% 38% 45% 41% 65% 39% ref|YP_001245556. aureus subsp. aureus JH9 gi|227898330| Staphylococcus 324 33% 44% 44% 35% 39% 45% 41% 65% 40% ref|ZP_04016135.1 aureus subsp. aureus TCH60 gi|49482430| Staphylococcus 405 33% 44% 44% 35% 39% 45% 41% 65% 39% ref|YP_039654.1| aureus subsp. aureus MRSA252 gi|221138844| Staphylococcus 314 33% 44% 44% 35% 39% 45% 41% 65% 40% ref|ZP_03563646.1 aureus subsp. aureus str. JKD6008 gi|227556295| Staphylococcus 322 33% 44% 44% 35% 39% 45% 41% 65% 40% ref|ZP_03986342.1 aureus subsp. aureus MN8 gi|282915516| Staphylococcus 383 34% 44% 43% 36% 38% 45% 41% 65% 39% ref|ZP_06323288.1 aureus subsp. aureus D139 gi|283767927| Staphylococcus 385 34% 44% 43% 35% 38% 45% 41% 65% 39% ref|ZP_06340842.1 aureus subsp. aureus H19 gi|82749898| Staphylococcus 414 34% 44% 44% 35% 38% 45% 41% 65% 39% ref|YP_415639.1| aureus RF122 gi|258424299| Staphylococcus 368 34% 44% 44% 35% 38% 45% 41% 66% 39% ref|ZP_05687180.1 aureus A9635 gi|228475763| Staphylococcus 325 33% 45% 45% 35% 38% 47% 41% 66% 41% ref|ZP_04060481.1 hominis SK119 gi|70725377| Staphylococcus 412 33% 44% 45% 34% 37% 48% 42% 67% 41% ref|YP_252291.1| haemolyticus JCSC1435 gi|224477650| Staphylococcus 317 36% 46% 44% 37% 39% 47% 42% 64% 40% ref|YP_002635256. carnosus subsp. carnosus TM300 gi|73661481| Staphylococcus 413 34% 44% 45% 34% 38% 47% 42% 62% 38% ref|YP_300262.1| saprophyticus subsp. saprophyticus ATCC 15305 gi|222151578| Macrococcus 61 36% 45% 44% 36% 37% 48% 44% 100% 41% ref|YP_002560734. caseolyticus JCSC5402 gi|281491840| Lactococcus 382 36% 45% 44% 37% 39% 44% 43% 46% 38% ref|YP_003353820. lactis subsp. lactis KF147 gi|51870502| Lactococcus 407 36% 45% 44% 38% 39% 44% 42% 46% 37% emb|CAG34226.1| lactis subsp. lactis gi|15673286| Lactococcus 272 30% 37% 36% 31% 31% 36% 35% 37% 31% ref|NP_267460.1| lactis subsp. lactis I11403 gi|44921617| Lactococcus 402 37% 45% 45% 39% 38% 44% 44% 45% 38% gb|AAS49166.1| lactis gi|229556973| Listeria grayi 52 35% 100% 44% 37% 39% 44% 40% 45% 38% ref|ZP_04444762.1 DSM 20601 gi|157369235| Serratia 275 31% 36% 40% 33% 32% 42% 38% 34% 36% ref|YP_001477224. proteamaculans 568 gi|270263480| Serratia 380 31% 36% 40% 33% 32% 41% 38% 34% 36% ref|ZP_06191749.1 odorifera 4Rx13 gi|293392629| Serratia 393 31% 36% 40% 33% 31% 40% 37% 35% 35% ref|ZP_06636949.1 odorifera DSM 4582 gi|255318365| Acinetobacter 367 35% 36% 42% 36% 33% 40% 38% 37% 35% ref|ZP_05359598.1 radioresistens SK82 gi|262378595| Acinetobacter 378 35% 36% 42% 36% 33% 40% 38% 37% 35% ref|ZP_06071752.1 radioresistens SH164 gi|126642486| Acinetobacter 261 27% 30% 35% 28% 27% 33% 29% 30% 28% ref|YP_001085470. baumannii ATCC 17978 gi|215482721| Acinetobacter 310 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|YP_002324919. baumannii AB307-0294 gi|260556653| Acinetobacter 373 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|ZP_05828871.1 baumannii ATCC 19606 gi|184158976| Acinetobacter 295 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|YP_001847315. baumannii ACICU gi|169795173| Acinetobacter 292 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|YP_001712966. baumannii AYE gi|239501136| Acinetobacter 357 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|ZP_04660446.1 baumannii AB900 gi|293609760| Acinetobacter 395 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|ZP_06692062.1 sp. SH024 gi|260549701| Acinetobacter 372 33% 36% 42% 34% 33% 40% 37% 36% 34% ref|ZP_05823918.1 sp. RUH2624 gi|169632867| Acinetobacter 291 32% 36% 41% 33% 32% 39% 37% 35% 33% ref|YP_001706603. baumannii SDF gi|262278264| Acinetobacter 377 33% 35% 42% 34% 33% 40% 37% 36% 34% ref|ZP_06056049.1 calcoaceticus RUH2202 gi|91779968| Burkholderia 415 35% 36% 38% 35% 34% 40% 36% 36% 33% ref|YP_555176.1| xenovorans LB400 gi|116695342| Ralstonia 256 36% 35% 37% 35% 34% 39% 36% 34% 32% ref|YP_840918.1| eutropha H16 gi|227506048| Corynebacterium 321 35% 38% 39% 36% 33% 38% 37% 37% 37% ref|ZP_03936097.1 striatum ATCC 6940 gi|227831961| Corynebacterium 323 35% 38% 38% 37% 32% 37% 38% 36% 37% ref|YP_002833668. aurimucosum ATCC 700975 gi|157876137| Leishmania 277 39% 35% 36% 40% 35% 35% 34% 33% 32% ref|XP_001686429. major strain Friedlin gi|146099506| Leishmania 262 36% 33% 34% 38% 34% 33% 31% 32% 30% ref|XP_001468661. infantum JPCM5 gi|154336655| Leishmania 269 38% 34% 35% 39% 35% 35% 35% 34% 32% ref|XP_001564563. braziliensis MHOM/BR/75/M2904 gi|238895871| Klebsiella 355 43% 38% 43% 45% 42% 43% 41% 37% 35% ref|YP_002920607. pneumoniae NTUH-K2044 gi|262042017| Klebsiella 376 43% 38% 42% 45% 42% 42% 40% 37% 34% ref|ZP_06015197.1 pneumoniae subsp. rhinoscleromatis ATCC 13884 gi|152971277| Klebsiella 268 43% 38% 43% 45% 42% 42% 40% 37% 34% ref|YP_001336386. pneumoniae subsp. pneumoniae MGH 78578 gi|288934189| Klebsiella 388 43% 39% 42% 44% 43% 42% 41% 37% 34% ref|YP_003438248. variicola At- 22 gi|290508392| Klebsiella sp. 390 43% 39% 42% 44% 43% 42% 41% 37% 34% ref|ZP_06547763.1 1_1_55 gi|206579495| Klebsiella 305 42% 38% 42% 44% 42% 42% 40% 37% 34% ref|YP_002237253. pneumoniae 342 gi|237732391| Citrobacter 349 43% 38% 40% 44% 42% 42% 41% 36% 35% ref|ZP_04562872.1 sp. 30_2 gi|283832207| Citrobacter 387 44% 38% 42% 46% 42% 42% 41% 37% 34% ref|ZP_06351948.1 youngae ATCC 29220 gi|157144683| Citrobacter 274 44% 38% 40% 46% 42% 42% 41% 36% 35% ref|YP_001452002. koseri ATCC BAA-895 gi|283786472| Citrobacter 386 43% 40% 41% 45% 41% 41% 41% 35% 35% ref|YP_003366337. rodentium ICC168 gi|16761331| Salmonella 284 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|NP_456948.1| enterica subsp. enterica serovar Typhi str. CT18 gi|213583505| Salmonella 309 39% 35% 37% 41% 38% 37% 38% 33% 33% ref|ZP_03365331.1 enterica subsp. enterica serovar Typhi str. E98-0664 gi|161612850| Salmonella 282 43% 38% 41% 45% 41% 41% 41% 36% 36% ref|YP_001586815. enterica subsp. enterica serovar Paratyphi B str. SPB7 gi|16765731| Salmonella 285 43% 38% 41% 45% 41% 41% 41% 36% 36% ref|NP_461346.1| enterica subsp. enterica serovar Typhimurium str. LT2 gi|197247765| Salmonella 301 43% 38% 41% 45% 41% 41% 41% 36% 36% ref|YP_002147363. enterica subsp. enterica serovar Agona str. SL483 gi|168242551| Salmonella 288 43% 39% 41% 45% 41% 41% 41% 36% 36% ref|ZP_02667483.1 enterica subsp. enterica serovar Heidelberg str. SL486 gi|224583057| Salmonella 318 43% 38% 41% 45% 41% 41% 41% 36% 36% ref|YP_002636855. enterica subsp. enterica serovar Paratyphi C strain RKS4594 gi|205353519| Salmonella 304 43% 39% 41% 45% 41% 41% 41% 36% 36% ref|YP_002227320. enterica subsp. enterica serovar Gallinarum str. 287/91 gi|62180978| Salmonella 411 43% 38% 41% 45% 41% 41% 41% 36% 36% ref|YP_217395.1| enterica subsp. enterica serovar Choleraesuis str. SC-B67 gi|168261562| Salmonella 289 43% 39% 41% 45% 41% 41% 41% 36% 36% ref|ZP_02683535.1 enterica subsp. enterica serovar Hadar str. RI_05P066 gi|168817849| Salmonella 290 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|ZP_02829849.1 enterica subsp. enterica serovar Weltevreden str. HI_N05- 537 gi|200388821| Salmonella 302 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|ZP_03215433.1 enterica subsp. enterica serovar Virchow str. SL491 gi|204929204| Salmonella 303 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|ZP_03220347.1 enterica subsp. enterica serovar Javiana str. GA_MM04042433 gi|168237435| Salmonella 287 43% 38% 42% 45% 41% 41% 41% 35% 36% ref|ZP_02662493.1 enterica subsp. enterica serovar Schwarzengrund str. SL480 gi|238912825| Salmonella 356 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|ZP_04656662.1 enterica subsp. enterica serovar Tennessee str. CDC07-0191 gi|168229788| Salmonella 286 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|ZP_02654846.1 enterica subsp. enterica serovar Kentucky str. CDC 191 gi|56412697| Salmonella 409 43% 38% 41% 45% 41% 41% 41% 35% 36% ref|YP_149772.1| enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 gi|161502438| “Salmonella 281 42% 38% 41% 44% 41% 41% 41% 36% 36% ref|YP_001569550. enterica subsp. arizonae serovar 62:z4, z23:—” gi|118333|sp| Enterobacter 257 43% 36% 42% 45% 42% 43% 42% 37% 37% P23234|DCIP_ENTCL cloacae gi|18652678| Enterobacter 296 43% 36% 42% 45% 42% 43% 42% 37% 37% gb|AAG00523.2| cloacae AF28 gi|295058524| Enterobacter 396 42% 37% 42% 44% 43% 42% 43% 37% 38% gb|ADF63262.1| cloacae subsp. cloacae ATCC 13047 gi|261340738| Enterobacter 375 42% 37% 41% 44% 41% 42% 43% 37% 37% ref|ZP_05968596.1 cancerogenus ATCC 35316 gi|295098023| Enterobacter 397 43% 37% 41% 45% 42% 42% 43% 37% 37% emb|CBK87113.1| cloacae NCTC 9394 gi|146312564| Enterobacter 263 43% 36% 40% 45% 43% 41% 41% 37% 37% ref|YP_001177638. sp. 638 gi|156933042| Cronobacter 273 45% 36% 40% 47% 42% 41% 40% 34% 34% ref|YP_001436958. sakazakii ATCC BAA-894 gi|260598789| Cronobacter 374 44% 36% 39% 46% 42% 41% 40% 34% 34% ref|YP_003211360. turicensis z3032 gi|291618298| Pantoea 391 42% 37% 41% 43% 41% 43% 37% 37% 33% ref|YP_003521040. ananatis LMG 20103 gi|1507711| Pantoea 267 44% 40% 42% 45% 43% 44% 41% 38% 35% gb|AAB06571.1| agglomerans gi|258634877| Pantoea sp. 369 45% 38% 42% 47% 43% 43% 41% 38% 36% ref|ZP_05727642.1 At-9b gi|292488940| Erwinia 392 46% 38% 43% 48% 43% 44% 39% 36% 34% ref|YP_003531827. amylovora CFBP1430 gi|259907830| Erwinia 371 46% 38% 43% 48% 43% 44% 39% 36% 35% ref|YP_002648186. pyrifoliae Ep1/96 gi|188533259| Erwinia 298 46% 37% 42% 48% 44% 44% 41% 35% 35% ref|YP_001907056. tasmaniensis Et1/99 gi|50122526| Pectobacterium 406 44% 40% 43% 44% 42% 44% 42% 39% 35% ref|YP_051693.1| atrosepticum SCRI1043 gi|227112359| Pectobacterium 319 44% 40% 42% 44% 42% 45% 42% 39% 36% ref|ZP_03826015.1 carotovorum subsp. brasiliensis PBR1692 gi|253689788| Pectobacterium 362 44% 40% 43% 44% 42% 45% 41% 38% 35% ref|YP_003018978. carotovorum subsp. carotovorum PC1 gi|270262920| Serratia 379 44% 38% 41% 44% 41% 43% 38% 37% 35% ref|ZP_06191191.1 odorifera 4Rx13 gi|157371649| Serratia 276 43% 40% 42% 46% 43% 44% 39% 39% 36% ref|YP_001479638. proteamaculans 568 gi|293395069| Serratia 394 44% 40% 44% 45% 44% 46% 41% 40% 35% ref|ZP_06639356.1 odorifera DSM 4582 gi|156538983| Nasonia 271 37% 36% 39% 38% 37% 39% 37% 35% 33% ref|XP_001600823. vitripennis gi|123441559| Yersinia 260 43% 39% 43% 45% 41% 44% 40% 36% 35% ref|YP_001005545. enterocolitica subsp. enterocolitica 8081 gi|238799418| Yersinia 354 43% 40% 43% 44% 42% 45% 40% 37% 36% ref|ZP_04642848.1 mollaretii ATCC 43969 gi|238764156| Yersinia 353 43% 39% 43% 44% 40% 43% 39% 38% 34% ref|ZP_04625109.1 kristensenii ATCC 33638 gi|238753136| Yersinia 351 43% 40% 43% 44% 40% 44% 40% 37% 37% ref|ZP_04614583.1 rohdei ATCC 43380 gi|238759526| Yersinia 352 43% 39% 43% 44% 41% 44% 40% 37% 35% ref|ZP_04620689.1 aldovae ATCC 35236 gi|189028459| Mycobacterium 299 83% 37% 40% 99% 47% 42% 41% 36% 35% sp|A0QBE6| avium 104 KDC_MYC gi|118464281| Mycobacterium 55 83% 37% 40% 100% 47% 42% 41% 36% 35% ref|YP_880234.1| avium 104 gi|254773861| Mycobacterium 365 82% 37% 40% 99% 48% 42% 41% 36% 35% ref|ZP_05215377.1 avium subsp. avium ATCC 25291 gi|41406881| Mycobacterium 400 82% 37% 40% 99% 48% 42% 41% 36% 35% ref|NP_959717.1| avium subsp. paratuberculosis K-10 gi|254818314| Mycobacterium 366 83% 36% 39% 92% 47% 41% 41% 36% 34% ref|ZP_05223315.1 intracellulare ATCC 13950 gi|118616174| Mycobacterium 259 83% 36% 38% 84% 46% 40% 40% 36% 34% ref|YP_904506.1| ulcerans Agy99 gi|183984651| Mycobacterium 294 83% 36% 38% 84% 46% 40% 40% 36% 34% ref|YP_001852942. marinum M gi|5840266| Mycobacterium 279 83% 36% 38% 85% 46% 41% 41% 37% 35% ref|NP_335303.1| tuberculosis CDC1551 gi|15607993| Mycobacterium 270 83% 37% 39% 85% 46% 42% 41% 37% 35% ref|NP_215368.1| tuberculosis H37Rv gi|240171442| Mycobacterium 51 100% 35% 37% 83% 46% 40% 39% 36% 34% ref|ZP_04750101.1 kansasii ATCC 12478 gi|15828161| Mycobacterium 278 73% 34% 39% 75% 45% 40% 39% 37% 34% ref|NP_302424.1| leprae TN gi|118470767| Mycobacterium 258 73% 35% 40% 75% 45% 44% 41% 37% 36% ref|YP_889968.1| smegmatis str. MC2 155 gi|237786605| Corynebacterium 350 53% 39% 40% 55% 49% 41% 40% 39% 36% ref|YP_002907310. kroppenstedtii DSM 44385 gi|258654904| Nakamurella 370 51% 36% 38% 53% 43% 37% 38% 34% 33% ref|YP_003204060. multipartita DSM 44233 gi|93005792| Psychrobacter 416 46% 39% 39% 46% 93% 40% 38% 37% 35% ref|YP_580229.1| cryohalolentis K5 gi|71065418| Psychrobacter 56 46% 39% 39% 47% 100% 41% 39% 37% 35% ref|YP_264145.1| arcticus 273-4 gi|148654159| Psychrobacter 265 47% 39% 39% 48% 74% 41% 39% 38% 35% ref|YP_001281252. sp. PRwf-1 gi|227504494| Corynebacterium 320 49% 37% 38% 48% 46% 40% 38% 36% 35% ref|ZP_03934543.1 striatum ATCC 6940

Preparation of Profile HMM

The amino acid sequences of the 170 sequences described above and provided in Table Z were analyzed using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMER software program is a profile Hidden Markov Model (profile HMM) that characterizes the input sequences. As stated in the user guide, profile HMMs are statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids (or nucleotides) and position specific scores for opening and extending an insertion or deletion. Compared to other profile based methods, HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus. Ashburn, Va.).

The profile HMM was built as follows:

Step 1. Build a Sequence Alignment

The 170 sequences in the cluster as described above were aligned using Clustal W (Thompson, J. D., Higgins, D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673 4680) with default parameters.

Step 2. Build a Profile HMM

The hmmbuild program was run on the set of aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new profile HMM, and saves the profile HMM to file. Using this program an un-calibrated profile was generated from the multiple sequence alignment of the 170 sequences as described in Step 1.

The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a profile HMM. A profile HMM is a linear state machine consisting of a series of nodes, each of which corresponds roughly to a position (column) in the multiple sequence alignment from which it is built. If gaps are ignored, the correspondence is exact, i.e., the profile HMM has a node for each column in the alignment, and each node can exist in one state, a match state. The word “match” here implies that there is a position in the model for every position in the sequence to be aligned to the model. Gaps are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”.

A profile HMM has several types of probabilities associated with it. One type is the transition probability—the probability of transitioning from one state to another. There are also emissions probabilities associated with each match state, based on the probability of a given residue existing at that position in the alignment. For example, for a fairly well-conserved column in an alignment, the emissions probability for the most common amino acid may be 0.81, while for each of the other 19 amino acids it may be 0.01.

A profile HMM is completely described in a HMMER2 profile save file, which contains all the probabilities that are used to parameterize the HMM. The emission probabilities of a match state or an insert state are stored as log-odds ratio relative to a null model:log₂ (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24. State transition scores are also stored as log odds parameters and are proportional to log₂(t_x). Where t_x is the transition probability of transiting from one state to another state.

Step 3. Calibrate the Profile HMM

The profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ□ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the profile HMM.

The calibrated profile HMM for the set of KIVD sequences is provided in Table Z and is herein referred to as the KIVD cluster profile HMM. In the main model section starting from the HMM flag line, the model has three lines per node, for M nodes (where M is the number of match states, as given by the LENG line). The first line reports the match emission log-odds scores:the log-odds ratio of emitting each amino acid from that state and from the Null model. The first number if the node number (1 . . . M). The next K numbers for match emission scores, one per amino acid. The highest scoring amino acid is indicated in the parenthesis after the node number. These log-odds scores can be converted back to HMM probabilities using the null model probability. The last number on the line represents the alignment column index for this match state. The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E.

Step 4. Test the Specificity and Sensitivity of the Built Profile HMMs

The Profile HMM was evaluated using hmmsearch, which reads a Profile HMM from hmmfile and searches a sequence file for significantly similar sequence matches. The sequence file searched contained 601 sequences (see above). During the search, the size of the database (Z parameter) was set to 1 billion. This size setting ensures that significant E-values against the current database will remain significant in the foreseeable future. The E-value cutoff was set at 10.

A hmmer search with the KIVD cluster profile HMM (Table Z, incorporated herein by reference) generated from the alignment of the 170 KIVD homologs, matched all 601 sequences with an E value<1E-223. This result indicates that members of the KIVD cluster share significant sequence similarity. A hmmer search with a cutoff of E value E-223 was used to separate KIVDs from other proteins. Thus, it is believed that polypeptides having a KIVD cluster profile HMM E value<1E-223 using hmmer search are suitable candidates for embodiments provided herein, such as for methods and recombinant host cells comprising the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde.

It will be appreciated that, equipped with this disclosure and using a combination of structural and sequence information available in the art and provided herein, polypeptides comprising ketoisovalerate decarboxylase activity and less than 100% identity to the exemplified sequences in Tables 4, 5 and 6 may be constructed for use in host cells and methods provided herein. For example, because Applicants have demonstrated that the polypeptides provided herein have ketoisovalerate decarboxylase activity, one of skill in the art may utilize biochemical, molecular, and structural information provided herein by way of sequence information and Profile HMM as well as information available in the art regarding ketoisovalerate decarboxylases. For example, Berthold, et al., provide the structure of holo-KdcA from L. lactis (Acta Crys. (2007) D63: 1217-1224); Yep, et al. (Bioorganic Chemistry, 34 (2006) 325-336) developed a homology model of the structure for KdcA from L. lactis, identifying residues Ser286, Phe381, Val461, and Met358 as residues that appeared to shape the substrate binding pocket; Smit, et al. (Appl. Environ. Microbiol. (2005) 71:303-311) aligned the amino acid sequence of KdcA with two decarboxylating enzymes, indolepyruvate decarboxylase of Enterobacter cloacae, and yeast pyruvate decarboxylase, which have been studied by X-ray crystallography.

One of ordinary skill in the art, equipped with this disclosure, can also generate active fragments of polypeptides provided herein, for example, by truncating polypeptides provided herein based on sequence alignments at the N-terminus or C-terminus and confirming α-ketoisovalerate decarboxylase activity.

Accordingly, polypeptides capable of catalyzing the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde disclosed herein include, but are not limited to, polypeptides comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to any one of SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, or 63 or an active fragment thereof. In embodiments, the polypeptide has a KIVD duster profile HMM E value of less than 1E-223 using the hmmsearch program. Similarly, Applicants provide herein polynucleotides encoding such polypeptides which include, but are not limited to polynucleotides comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to any one of SEQ ID NO: 30, 31, 32, 34, 35, 37, 38, 40, or 42, or sequences thereof which are codon-optimized for expression in a particular host cell such as, for example, E. coli or S. cerevisiae.

Thiamine diphosphate (also known as thiamine pyrophosphate or “TPP” or “TDP”) is a coenzyme employed by KIVD as a cofactor. Thiamine is the vitamin form, which may be supplemented in reaction medium such as fermentation medium. Once transported into a cell, thiamine will be converted to thiamine diphosphate. In addition, certain polypeptides having ketoisovalerate decarboxylase activity disclosed herein have increased affinity for TPP as compared to the Lactococcus lactis polypeptide having SEQ ID NO: 68, and, as such, may provide advantages. For example, polypeptides provided herein may be useful to catalyze the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde under conditions of low or diminished thiamine, potentially reducing costs and protocol complexity. In embodiments, such polypeptides comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to any one of SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, or 63 or an active fragment thereof. In some embodiments, such polypeptides comprise the sequences of kivD81, Mca, or kdcA from Listeria grayi, Macrococcus caseolyticus, or Lactococcus lactis, respectively, or an active fragment thereof. In some embodiments, such polypeptides comprise at least about 80% identity to SEQ ID NO: 52, 61, or 66 or an active fragment thereof. In some embodiments, such polypeptides comprise at least about 80% identity to SEQ ID NO: 52 or 61 or an active fragment thereof.

Polypeptides and the polynucleotides encoding them provided herein are also useful for expression in recombinant host cells, such as recombinant host cells comprising an isobutanol biosynthetic pathway which includes the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde. Furthermore, Applicants have shown that polypeptides provided herein are at least as capable of converting α-ketoisovalerate to isobutyraldehyde as a Lactococcus lactis polypeptide sequence having SEQ ID NO: 68 in a recombinant host cell, as measured by α-ketoisovalerate accumulation during a fermentation (See Example 12). Similarly, polypeptides provided herein can provide increased isobutanol yield in recombinant host cells as compared to the isobutanol yield observed in cells employing a Lactococcus lactis polypeptide sequence having SEQ ID NO: 68 (See Example 12). Therefore, provided herein are recombinant host cells comprising polypeptides comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to any one of SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, or 63 or an active fragment thereof, or a polynucleotide encoding such a polypeptide.

Construction of Recombinant Host Cells

The genetic manipulations of the host cells described herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202). In embodiments, the recombinant host cells disclosed herein can be any bacteria, yeast or fungi host useful for genetic modification and recombinant gene expression. In other embodiments, a recombinant host cell can be a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In other embodiments, the host cell can be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Candida glabrata, Candida albicans, Pichia stipitis, Yarrowia lipolytica, E. coli, or L. plantarum. In still other embodiments, the host cell is a yeast host cell. In some embodiments, the host cell is a member of the genera Saccharomyces. In some embodiments, the host cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe. In some embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

Methods for gene expression in recombinant host cells, including, but not limited to, yeast cells are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). In embodiments, the coding region for the α-ketoisovalerate decarboxylase enzymes to be expressed can be codon optimized for the target host cell, as well known to one skilled in the art. Expression of genes in recombinant host cells, including but not limited to yeast cells, can require a promoter operably linked to a coding region of interest, and a transcriptional terminator. A number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, and GPM1; and the following inducible promoters suitable for use in yeast: GAL1, GAL10 and CUP1. Other yeast promoters include hybrid promoters UAS(PGK1)-FBA1p (SEQ ID NO: 228), UAS(PGK1)-ENO2p (SEQ ID NO: 229), UAS(FBA1)-PDC1p (SEQ ID NO: 230), UAS(PGK1)-PDC1p (SEQ ID NO: 231), and UAS(PGK)-OLE1p (SEQ ID NO: 232). Suitable transcriptional terminators that can be used in a chimeric gene construct for expression include, but are not limited to, FBA1t, TDH3t, GPM1t, ERG10t, GAL1t, CYC1t, and ADH1t.

Vectors useful for the transformation of a variety of host cells are common and described in the literature. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors can comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.

In embodiments, suitable promoters, transcriptional terminators, and α-ketoisovalerate decarboxylase coding regions can be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells. Such vectors allow strain propagation in both E. coli and yeast strains, and can contain a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast include, but are not limited to, shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2-micron origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are HIS3 (vector pRS423), TRP1 (vector pRS424), LEU2 (vector pRS425) and URA3 (vector pRS426).

In embodiments, construction of expression vectors with a chimeric gene encoding the described enzymes can be performed by the gap repair recombination method in yeast. In embodiments, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain an approximately 21 bp sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X,” a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g. TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. In embodiments, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding region X-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.

KIVD Activity

The presence of α-ketoisovalerate decarboxylase activity in the recombinant host cells disclosed herein can be confirmed using routine methods known in the art. In a non-limiting example, and as described in the Examples herein, transformants can be screened by PCR using primers for α-ketoisovalerate decarboxylase genes. In another non-limiting example, and as described in the examples herein, α-ketoisovalerate decarboxylase activity can be assayed by expressing α-ketoisovalerate decarboxylase enzymes disclosed herein in a recombinant host cell disclosed herein that lacks endogenous α-ketoisovalerate decarboxylase activity. In another non-limiting example, α-ketoisovalerate decarboxylase activity can be confirmed by more indirect methods, such as by measuring the products downstream from isobutyraldehyde in a biosynthetic pathway. In another non-limiting example, α-ketoisovalerate decarboxylase activity can be confirmed by measuring cofactor disappearance, or by measuring cofactor disappearance for a step downstream from isobutyraldehyde in a biosynthetic pathway, for example, a coupled assay as described herein and/or in the art (see, for example, Zhang et al., PNAS (2008) 105(52): 20653-20658). In another non-limiting example, α-ketoisovalerate decarboxylase activity can be confirmed by determining the rates for the conversion of α-ketoisovalerate to isobutryaldehyde with the aldehyde-specific Purpald® colorimetric enzyme assay, described herein. In another non-limiting example, α-ketoisovalerate decarboxylase activity can be confirmed by measuring disappearance of α-ketoisovalerate using methods known in the art (see, for example, de la Plaza, et al. FEMS Microbiol Letters (2004) 238:367-374).

The TPP cofactor activation constant, which provides a measure of the enzyme affinity for TPP is described in Chang et al. (Biochem. J. (1999) 339: 255-260). The TPP cofactor activation constant of polypeptides provided herein can be determined by plotting the specific activities observed as a function of TPP concentration. The rates for the conversion of α-ketoisovalerate to isobutyraldehyde may be measured in a horse liver ADH (hADH) coupled enzyme assay, which has been described in Gocke, D. et al. (Adv. Synth. Catal. 2007, 349, 1425-1435), run at different TPP concentrations. Specific activities are then plotted against TPP concentration. Resulting curves may then be fit to the saturation equation SA=(SA_(max)*[TPP])/(K_(c)+[TPP])+C, where SA is the specific activity, SA_(max) is maximum specific activity, K_(c) is the cofactor activation constant, [TPP] is the concentration of TPP, and C is the activity in the absence of added TPP, using software such as Kaleidagraph (Synergy) to determine the cofactor activation constant (K_(c)). The TPP affinity of polypeptides can also be measured via fluorescence quenching as described in Chang et al. (Biochem. J. (1999) 339:255-260).

In embodiments, polypeptides provided herein have a TPP cofactor activation constant (K_(c)) less than about 5 μM, less than about 10 μM, less than about 20 μM, less than about 30 μM, less than about 40 μM, less than about 50 μM, less than about 70 μM, or less than about 90 μM.

The substrate specificity ratio provides a measure of the ability of an enzyme to discriminate between the reactions of competing substrates and is described by Fersht, A. (Enzyme Structure and Mechanism, 1977, 1985 W.H. Freeman and Company, New York). The α-ketoisovalerate to pyruvate specificity ratio for a given polypeptide can be determined by measuring the V_(max) and K_(M) values for the conversion of α-ketoisovalerate to isobutyraldehyde and the V_(max) and K_(M) values for the conversion of pyruvate to acetaldehyde. The rates for the conversion of α-ketoisovalerate to isobutyraldehyde and for pyruvate to acetaldehyde may be measured in a horse liver ADH (hADH) coupled enzyme assay which has been described in Gocke, D. et al. (Adv. Synth. Catal. 2007, 349, 1425-1435) run at different substrate concentration. Specific activities are then plotted against substrate concentration. Resulting curves may then be fit to the Michaelis-Menton using software such as Kaleidagraph (Synergy) to determine V_(max) and K_(M) values for each substrate. “Specificity ratio” as used herein is the quotient of V_(max)/K_(M) determined for α-ketoisovalerate divided by the V_(max)/K_(M) determined for pyruvate.

In embodiments, the specificity ratio for polypeptides disclosed herein is greater than about 1, greater than about 10, greater than about 20, greater than about 50, greater than about 100, greater than about 150, greater than about 200, greater than about 250, or greater than about 300.

Provided herein are methods of converting α-ketoisovalerate to isobutyraldehyde employing the polypeptides disclosed. In embodiments, methods comprise: (a) providing a polypeptide wherein said polypeptide comprises at least one of: (i) at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof and α-ketoacid decarboxylase activity; or (ii) α-ketoacid decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than about 1, greater than about 10, greater than about 100, greater than about 150, or greater than about 200, and thiamine diphosphate cofactor activation constant (K_(c)) of about 100 μM, about 50 μM, or about 20 μM or less; and (b) contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced. In embodiments, methods comprise: (a) providing a polypeptide wherein said polypeptide comprises at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof and α-ketoacid decarboxylase activity; and (b) contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced. In embodiments, said polypeptide comprises at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 52 or 61 or an active fragment thereof. In embodiments, methods comprise: (a) providing a polypeptide wherein said polypeptide comprises α-ketoacid decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than about 1, greater than about 10, greater than about 100, greater than about 150, or greater than about 200, and thiamine diphosphate cofactor activation constant (K_(c)) of about 100 μM, about 50 μM, or about 20 μM or less; and (b) contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced. In embodiments, the contacting occurs in the presence of less than about 10 mg/L thiamine. In embodiments, the contacting occurs in the presence of less than about 30 mg/L thiamine, less than about 20 mg/L thiamine, or less than about 5 mg/L thiamine. In embodiments, the contacting occurs in the presence of about 1 to about 10 mg/L thiamine, about 5 to about 20 mg/L thiamine, about 10 to about 15 mg/L thiamine, about 5 to about 15 mg/L thiamine, about 5 to about 10 mg/L thiamine, or about 1 to about 5 mg/L thiamine. In embodiments, the contacting occurs within a recombinant host cell and wherein the polypeptide is heterologous to recombinant host cell. In embodiments, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In embodiments, the recombinant host cell is Saccharomyces cerevisiae. In embodiments, the recombinant host cell further comprises heterologous polynucleotides encoding polypeptides which catalyze the substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; and (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In embodiments, the host cell further comprises a heterologous polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol. In embodiments, the recombinant host cell further comprises reduced or eliminated pyruvate decarboxylase activity. In embodiments, the recombinant host cell further comprises at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis. In embodiments, the recombinant host cell comprises deletion of fra2. In embodiments, the recombinant host cell comprises reduced or eliminated glycerol-3-phosphate dehydrogenase activity.

Production of Isobutanol

Provided herein are methods of producing isobutanol. In embodiments, the methods comprise: (a) providing a host cell comprising an isobutanol biosynthetic pathway and a polypeptide wherein said polypeptide comprises at least one of: i) at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61, or 63 or an active fragment thereof and α-ketoacid decarboxylase activity or (ii) α-ketoacid decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than about 1, greater than about 10, greater than about 100, greater than about 150, or greater than about 200, and thiamine diphosphate cofactor activation constant (K_(c)) of about 100 μM, about 50 μM, or about 20 μM or less; and (b) contacting the host cell with a fermentable carbon substrate under conditions whereby isobutanol is produced. In embodiments, the contacting occurs in the presence of less than about 10 mg/L thiamine or less than about 1 mg/L thiamine. In embodiments, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In embodiments, the recombinant host cell is Saccharomyces cerevisiae. In embodiments, the recombinant host cell further comprises heterologous polynucleotides encoding polypeptides which catalyze the substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; and (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In embodiments, the host cell further comprises a heterologous polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol. In embodiments, the recombinant host cell further comprises reduced or eliminated pyruvate decarboxylase activity. In embodiments, the recombinant host cell further comprises at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis. In embodiments, the recombinant host cell further comprises deletion of fra2. In embodiments, the recombinant host cell comprises reduced or eliminated glycerol-3-phosphate dehydrogenase activity. In embodiments isobutanol is produced at an effective yield greater than that produced by the analogous host cell comprising a heterologous polynucleotide encoding a polypeptide comprising SEQ ID NO: 68 and not (i) or (ii).

Accordingly, also provided herein are recombinant host cells comprising (i) a heterologous polypeptide which catalyzes the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde wherein said polypeptide comprises at least about 80% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof; or a heterologous polynucleotide encoding a heterologous polypeptide of (i). In embodiments, said polypeptide comprises at least about 95% identity to SEQ ID NO: 51, 52, 53, 55, 56, 58, 59, 61 or 63 or an active fragment thereof. In embodiments, host cells further comprise heterologous polynucleotides encoding polypeptides which catalyze the substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In embodiments, host cells further comprising a heterologous polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol. In embodiments, each of the polypeptides which catalyze the substrate to product conversions are non-native to the host cell. In embodiments, the recombinant host cell comprises reduced or eliminated pyruvate decarboxylase activity. In embodiments, the recombinant host cell comprises at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis. In embodiments, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In embodiments, the recombinant host cell is Saccharomyces cerevisiae.

Isobutanol Biosynthetic Pathway

Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Pat. Nos. 7,851,188, 7,889,993, and 8,178,328, all incorporated herein by reference. One isobutanol biosynthetic pathway comprises the following substrate to product conversions:

pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase;

α-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for example, by a branched-chain keto acid decarboxylase; and

isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

In some embodiments, the isobutanol biosynthetic pathway comprises at least one polynucleotide, at least two polynucleotides, at least three polynucleotides, or at least four polynucleotides that is/are heterologous to the host cell. In embodiments, each substrate to product conversion of an isobutanol biosynthetic pathway in a recombinant host cell is catalyzed by a heterologous polypeptide. In embodiments, the polypeptide catalyzing the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol are capable of utilizing NADH as a cofactor.

Genes and polypeptides that can be used for substrate to product conversions described herein as well as methods of identifying such genes and polypeptides, are described herein and/or in the art, for example, for isobutanol, in the Examples and in U.S. Pat. Nos. 7,851,188, 7,889,993, and 8,178,328. Example ketol-acid reductoisomerase (KARI) enzymes are described in U.S. Pat. Nos. 8,129,162 and 8,222,017 and in U.S. Patent Appl. Pub. Nos. 20080261230 A1, 20100197519 A1, and PCT Appl. Pub. No. WO/2011/041415, all of which are incorporated by reference. Examples of KARIs disclosed therein are those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, and Pseudomonas fluorescens PF5 variants. KARIs include Anaerostipes caccae KARI variants “K9G9” and “K9D3” (SEQ ID NOs: 235 and 234, respectively). US Appl. Pub. No. 20100081154 A1, and U.S. Pat. No. 7,851,188 describe dihydroxyacid dehydratases (DHADs), including a DHAD from Streptococcus mutans (protein SEQ ID NO: 417). U.S. Pat. No. 8,188,250, incorporated herein by reference, describes SadB, an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol dehydrogenases also include horse liver ADH (“HADH”) and Beijerinkia indica ADH (protein SEQ ID NO: 247) described in US Appl. Pub. No. 20110269199, incorporated herein by reference.

It will be appreciated that host cells comprising an isobutanol biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Appl. Pub. No. 20090305363 (incorporated by reference) discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. Modifications to reduce glycerol-3-phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Patent Appl. Pub. No. 20090305363 (incorporated herein by reference), modifications to a host cell that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in U.S. Patent Appl. Pub. No. 20100120105 (incorporated herein by reference). Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway. Other modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In embodiments, the polypeptide having acetolactate reductase activity is YMR226C (SEQ ID NO: 236) of Saccharomyces cerevisae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 (SEQ ID NO: 233) from Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Appl. Publication No. 20110124060, incorporated herein by reference.

Recombinant host cells may further comprise (a) at least one heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity; and (b)(i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis; and/or (ii) at least one heterologous polynucleotide encoding a polypeptide affecting Fe—S cluster biosynthesis. In embodiments, the polypeptide affecting Fe—S cluster biosynthesis is encoded by AFT1 (nucleic acid SEQ ID NO: 237, amino acid SEQ ID NO: 238), AFT2 (SEQ ID NOs: 239 and 240), FRA2 (SEQ ID NOs: 241 and 242), GRX3 (SEQ ID NOs: 243 and 244), or CCC1 (SEQ ID NOs: 245 and 246). In embodiments, the polypeptide affecting Fe—S cluster biosynthesis is constitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT1 C293F.

Growth for Production

Recombinant host cells disclosed herein are grown in fermentation media which contains suitable carbon substrates. Additional carbon substrates may include, but are not limited to, monosaccharides such as fructose, oligosaccharides such as lactose, maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate, or glycerol.

Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, in some embodiments, the carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. 20070031918 A1. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, rapeseed, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers (e.g., sunflowers and safflower), animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of an enzymatic pathway described herein.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention include common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, Yeast Medium (YM) broth, or broth that includes yeast nitrogen base, ammonium sulfate, and dextrose (as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between about pH 5.0 to about pH 9.0. In one embodiment, about pH 6.0 to about pH 8.0 is used for the initial condition. Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 is used for the initial condition. Suitable pH ranges for the fermentation of other microorganisms are between about pH 3.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 is used for the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentations.

Industrial Batch and Continuous Fermentations

Isobutanol, or other products, may be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992).

Isobutanol, or other products, may also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the production of isobutanol, or other products, may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.

Methods for Isobutanol Isolation from the Fermentation Medium

Bioproduced isobutanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the isobutanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

Because isobutanol forms a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase may be further purified by distillation in a second distillation column.

The isobutanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the butanol from the solvent.

Distillation in combination with adsorption can also be used to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation may be used to isolate and purify the isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).

In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields. One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction. In general, with regard to butanol fermentation, for example, the fermentation medium, which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the fermentation medium form a biphasic mixture. The butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.

Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 2009/0305370, the disclosure of which is hereby incorporated in its entirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, and mixtures thereof. The extractant(s) for ISPR can be non-alcohol extractants. The ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.

In some embodiments, an ester can be formed by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst capable of esterifying the alcohol with the organic acid. In such embodiments, the organic acid can serve as an ISPR extractant into which the alcohol esters partition. The organic acid can be supplied to the fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol. The catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock. When the catalyst is supplied to the fermentation vessel, alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel. Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant. The extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration. In addition, unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.

In situ product removal can be carried out in a batch mode or a continuous mode. In a continuous mode of in situ product removal, product is continually removed from the reactor. In a batchwise mode of in situ product removal, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. For in situ product removal, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some embodiments of the present invention, the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel. The ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved. In some embodiments, the ester-containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.

Isobutanol titer in any phase can be determined by methods known in the art, such as via high performance liquid chromatography (HPLC) or gas chromatography, as described, for example in US20090305370, incorporated herein by reference.

EXAMPLES Plasmids

pYZO90 (SEQ ID NO: 69) was constructed to contain a chimeric gene having the coding region of the alsS gene from Bacillus subtilis (nt position 457-2172) expressed from the yeast CUP1 promoter (nt 2-449) and followed by the CYC1 terminator (nt 2181-2430) for expression of ALS, and a chimeric gene having the coding region of the ilvC gene from Lactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4682-5304) for expression of KARI.

pYZO90 was digested with SpeI and NotI to remove most of the CUP1 promoter and all of the alsS coding sequence and CYC terminator. The vector was then self-ligated after treatment with Klenow fragment and transformed into E. coli StbI3 cells, selecting for ampicillin resistance. Removal of the DNA region was confirmed for two independent clones by DNA sequencing across the ligation junction by PCR. The resulting plasmid was named pYZ090ΔalsS (SEQ ID NO: 182).

Plasmid pLH702 (SEQ ID NO: 218) was constructed in a series of steps from pYZ090 as described in the following paragraphs. This plasmid expresses KARI variant K9D3 (SEQ ID NO: 234) from the yeast ILV5 promoter.

pYZ058 (pHR81-PCUP1-AlsS-PILV5-yeast KARI) was derived from pYZO90 (pHR81-PCUP1-AlsS-PILV5-lactis KARI). pYZO90 was cut with PmeI and SfiI enzymes, and ligated with a PCR product of yeast KARI. The PCR product was amplified from genomic DNA of Saccharomyces cerevisiae BY4741 (Research Genetics Inc.) strain using upper primer 5′-catcatcacagtttaaacagtatgttgaagcaaatcaacttcggtgg-3′ (SEQ ID NO: 248) and lower primer 5′-ggacgggccctgcaggccttattggttttctggtctcaactttctgac-3′ (SEQ ID NO: 249), and digested with PmeI and SfiI enzymes. pYZ058 was confirmed by sequencing. pLH550 (pHR81-PCUP1-AlsS-PILV5-Pf5.KARI) was derived from pYZ058. The wild type Pf5.KARI gene was PCR amplified with OT1349 (5′-catcatcacagtttaaacagtatgaaagttttctacgataaagactgcgacc-3′ (SEQ ID NO: 250)) and OT1318 (5′-gcacttgataggcctgcagggccttagttcttggctttgtcgacgattttg-3′ (SEQ ID NO: 251)), digested with PmeI and SfiI enzymes and ligated with pYZ058 vector cut with PmeI and SfiI. The vector generated, pLH550, was confirmed by sequencing. pLH556 was derived from pLH550 by digesting the vector with SpeI and NotI enzymes, and ligating with a linker annealed from OT1383 (5′-ctagtcaccggtggc-3′ (SEQ ID NO: 252)) and OT1384 (5′-ggccgccaccggtga-3′ (SEQ ID NO: 253)) which contains overhang sequences for SpeI and NotI sites. This cloning step eliminates the alsS gene and a large fragment of the PCUP1 promoter from the plasmid, with 160 bp residual upstream sequence that is not functional. pLH556 was confirmed by sequencing. pLH702 was derived from pLH556. The K9D3 mutant KARI gene was excised from another vector using PmeI and SfiI enzymes, and ligated with pLH556 at PmeI and SfiI sites, replacing the Pf5.KARI gene with the K9D3 gene. The constructed vector pLH702 was confirmed by sequencing.

The pLH468 plasmid was constructed for expression of DHAD, KivD and HADH in yeast. pBP915 was constructed from pLH468 by deleting the kivD gene and 957 base pairs of the TDH3 promoter upstream of kivD. pLH468 was digested with SwaI and the large fragment (12896 bp) was purified on an agarose gel followed by a Gel Extraction kit (Qiagen; Valencia, Calif.). The isolated fragment of DNA was self-ligated with T4 DNA ligase and used to transform electrocompetent TOP10 Escherichia coli (Invitrogen; Carlsbad, Calif.). Plasmids from transformants were isolated and checked for the proper deletion by restriction analysis with the SwaI restriction enzyme. Isolates were also sequenced across the deletion site. A clone with the proper deletion was designated pBP915 (pLH468ΔkivD; SEQ ID NO: 166).

pYZ067 (SEQ ID NO: 254) was constructed to contain the following chimeric genes: 1) the coding region of the ilvD gene from S. mutans UA159 with a C-terminal Lumio tag expressed from the yeast FBA1 promoter followed by the FBA1 terminator for expression of dihydroxy acid dehydratase, 2) the coding region for horse liver ADH expressed from the yeast GPM1 promoter followed by the ADH1 terminator for expression of alcohol dehydrogenase, and 3) the coding region of the KivD gene from Lactococcus lactis expressed from the yeast TDH3 promoter followed by the TDH3 terminator for expression of ketoisovalerate decarboxylase.

Plasmid pYZ067ΔkivDΔhADH was constructed from pYZ067 by deleting the promoter-gene-terminator cassettes for both kivD and adh. pYZ067 was digested with BamHI and SacI (New England BioLabs; Ipswich, Mass.) and the 7934 bp fragment was purified on an agarose gel followed by a Gel Extraction kit (Qiagen; Valencia, Calif.). The isolated fragment of DNA was treated with DNA Polymerase I, Large (Klenow) Fragment (New England BioLabs; Ipswich, Mass.) and then self-ligated with T4 DNA ligase and used to transform competent TOP10 Escherichia coli (Invitrogen; Carlsbad, Calif.). Plasmids from transformants were isolated and checked for the proper deletion by sequence analysis. A correct plasmid isolate was designated pYZ067ΔkivDΔhADH (SEQ ID NO: 255).

Example 1 Expression of Candidate Enzymes in E. coli

Based on a biodiversity search (described herein above), genes encoding 9 enzymes (Table 5) were synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). Each gene was cloned under control of the T5 promoter in the vector pJexpress404 (DNA2.0, Menlo Park, Calif.) and expressed in E. coli Top10 (Invitrogen, San Diego, Calif.). Following shake flask growth at 37 C to OD600 nm=0.5 in LB media supplemented with 100 ug ampicillin/mL, with or without supplementation of 30 mg/L thiamine, 1 mM IPTG was added and the cultures grown for an additional 14-16 hrs. Cells were harvested by centrifugation and heterologous protein expression was confirmed by SDS-PAGE.

TABLE 5 Candidate KIVD enzymes. SEQ ID ID Organism source GI ref NO kivD75 Erwinia amylovora 292488940 optimized 2 NT native NT 25 AA 46 kivD76 Thauera sp. MZ1T 217968563 optimized 3 NT native NT 26 AA 47 kivD77 Frankia sp. Eul1c 280960373 optimized 4 NT native NT 27 AA 48 kivD78 Acinetobacter sp. 260549701 optimized 5 RUH2624 NT native NT 28 AA 49 kivD79 Anabaena variabilis 75910313 optimized 6 ATCC 29413 NT native NT 29 AA 50 kivD80 Mycobacterium kansasii 240171442 optimized 7 ATCC 12478 NT native NT 30 AA 51 kivD81 Listeria grayi DSM 20601 229556973 optimized 8 NT native NT 31 AA 52 kivD82 Bacillus thuringiensis 228908218 optimized 9 IBL 200 NT native NT 32 AA 53 kivD83 Staphylococcus epidermidis 242372336 optimized 10 M23864:W1 NT native NT 33 AA 54

Example 2 Expression of Candidate Enzymes in E. coli

Based on the enzyme assay results from the first round biodiversity search, genes encoding 13 additional enzymes (Table 6) were synthesized using codons optimized for expression in E. coli (DNA2.0, Menlo Park, Calif.). In addition, a codon optimized gene encoding the L. lactis kivD (control; SEQ ID NO:1) was prepared. Each gene was cloned under control of the T5 promoter in the vector pJexpress404 (DNA2.0, Menlo Park, Calif.) and expressed in E. coli Top10 (Invitrogen, San Diego, Calif.). Following shake flask growth at 37 C to OD600 nm=0.5 in LB media supplemented with 100 ug ampicillin/mL, with or without supplementation of 30 mg/L thiamine, 1 mM IPTG was added and the cultures grown for an additional 14-16 hrs. Cells were harvested by centrifugation and heterologous protein expression was confirmed by SDS-PAGE.

TABLE 6 Second round selection of candidate KIVD enzymes. SEQ ID ID Organism source GI ref NO Mav Mycobacterium 118464281 optimized 11 avium 104 NT native NT 34 AA 55 Par Psychrobacter 71065418 optimized 12 arcticus 273-4 NT native NT 35 AA 56 Cst Corynebacterium 227506048 optimized 13 striatum ATCC NT 6940 native NT 36 AA 57 Bme Bacillus megaterium 294497944 optimized 14 QM B1551 NT native NT 37 AA 58 Hmu Helicobacter 291276462 optimized 15 mustelae 12198 NT native NT 38 AA 59 Cac Clostridium 15004729 optimized 16 acetobutylicum NT ATCC 824 native NT 39 AA 60 Mca Macrococcus 222151578 optimized 17 caseolyticus NT JCSC5402 native NT 40 AA 61 Npu Nostoc punctiforme 186682481 optimized 18 PCC 73102 NT native NT 41 AA 62 Sve Sarcina ventriculi 16417060 optimized 19 NT native NT 42 AA 63 Bth Bacillus 228985570 optimized 20 thuringiensis NT serovar tochigiensis BGSC 4Y1 native NT 43 AA 64 Bce Bacillus cereus 229100258 optimized 21 Rock3-29 NT native NT 44 AA 65 kdcA Lactococcus lactis 44921617 optimized 22 NT native NT 45 AA 66 kdcA_F381W Lactococcus lactis optimized 23 NT AA 67 kivD Lactococcus lactis 51870502 optimized 1 (control) NT native NT 24 AA 68

Example 3 Identification of Enzymes that Exhibit KIVD Activity when Expressed in E. coli

Putative KIVD enzymes were identified as having the desired α-ketoisovalerate (αKIV) decarboxylase activity by enzymatic assay of E. coli cell free extracts, described below. The expression of the putative enzymes in E. coli is detailed in Examples 1 and 2.

Preparation of Cell Free Extract

E. coli cells were suspended in 3 mL of 100 mM HEPES, pH 6.8, 10 mM MgCl₂ and broken by sonication at 0° C. The cells were sonicated with a microtip probe for 10 seconds, with 25 seconds of rest. This cycle was repeated 12 times, for a total of 2 minutes of sonication. The crude extract from the broken cells was centrifuged to pellet the cell debris. The supernatants were removed and stored on ice until assayed.

Protein Quantification

The total protein concentration in cell free extracts was measured by the Bradford Assay using Coomassie Plus (Thermo Scientific #23238, Rockford, Ill.). BSA was employed as a standard. The concentration of protein was measured by following absorbance at 595 nm using a Cary 300 spectrophotometer (Agilent Technologies, Wilmington Del.).

KIVD Enzyme Assay Protocol—Coupled

The rates for the conversion of α-ketoisovalerate to isobutyraldehyde were measured in a horse liver ADH (hADH) coupled enzyme assay, which has been described in Gocke, D. et al. (Adv. Synth. Catal. 2007, 349, 1425-1435). Assays of cell free extracts were performed at 30° C. in buffer containing 100 mM HEPES pH 6.8, 10 mM MgCl₂, 200 μM NADH, 500 μM TPP, 30 mM α-KIV (Sigma), and 0.45 U hADH (1 mg=1.5 U) (Sigma). The oxidation of NADH was monitored at 340 nm in a 1 cm path length cuvette on a Cary 300 spectrophotometer (Agilent Technologies, Wilmington Del.) The enzyme rate was calculated using the molar extinction coefficient of 6220 M⁻¹ cm⁻¹ for NADH. Controls at various concentrations of hADH and cell free extract ensured that measured rate was determined by KIVD enzyme activity.

The specific activities of the putative KIVD enzymes expressed in E. coli were calculated from measured enzyme activities and total protein concentrations. The results from round 1 and 2 are shown in Tables 7 and 8, respectively.

TABLE 7 KIVD activities for Round 1 Putatives ID Specific Activity (U/mg) kivD75 0.1 kivD76 <0.1 kivD77 <0.1 kivD78 0.14 kivD79 <0.1 kivD80 4.0 kivD81 16.5 kivD82 1.6 kivD83 0.07

TABLE 8 KIVD activities for Round 2 Putatives ID Specific Activity (U/mg) Mav 1.2 Par 2.7 Cst <0.1 Bme <0.1 Hmu <0.1 Cac 0.4 Mca 13.9 Npu <0.1 Sve 0.23 Bth 1.9 Bce <0.1 kdcA 11.8 kivD 59.5 Vector control <0.1 KIVD Enzyme Assay Protocol—Colorimetric

The rates for the conversion of α-ketoisovalerate to isobutryaldehyde for three enzymes with high KIVD activities were also measured with the aldehyde-specific Purpald® (Sigma) colorimetric enzyme assay, which has been described by DuPont-Durst and Gokel (J. Chem Edu. 1978, 55, 206). Assays of cell free extracts were performed at 30° C. in buffer containing 100 mM HEPES pH 6.8, 10 mM MgCl₂, 500 μM TPP and 30 mM α-KIV. The total reaction volume was 1 mL. Formation of isobutyraldehyde was monitored by removing 200 μL aliquots at fixed time points (0, 10, 20 and 30 minutes) and quenching the reaction in 1 mL of Purpald® stock solution (5 mg/mL of Purpald in 2 M NaOH). The mixture was then incubated at room temperature for 20 minutes to allow for color development. The mixture was mixed every 5 minutes during the incubation process. A standard curve of 0 to 10 mM isobutyraldehyde was used to determine the concentration of isobutyraldehyde being generated by the enzyme. 200 μL of each standard was made and added to 1 mL Purpald® stock solution alongside the zero time point. The standard was incubated at room temperature for 20 minutes and mixed every 5 minutes. Absorbance at 535 nm was measured with a Cary 300 spectrophotometer (Agilent Technologies, Wilmington Del.). The specific activities of the putative KIVD enzymes expressed in E. coli were calculated from measured enzyme activities and total protein concentrations.

Equivalent specific activities were found with the colorimetric and continuous coupled assays as shown in Table 9.

TABLE 9 Comparison of Colorimetric and Coupled Assays SA, U/mg SA, U/mg ID (Colorimetric) (Coupled) KivD (L. lactis) 27.0 29.0 KivD81 5.9 6.3 Mca 14.9 17

Example 4 Measurement of TPP Cofactor Activation Constant of KIVD to Enzymes

The three putative KIVD enzymes with the highest activity from Rounds 1 and 2 (KivD80, KivD81, and Mca), L. lactis KivD and KdcA were evaluated for TPP cofactor activation constants in crude lysates of the E. coli cells. The expression of these enzymes in E. coli is described in Examples 1 and 2. Generation of crude lysates and protein quantification were performed as described in Example 3.

Desalting of Cell Free Extract

Zeba Desalt Spin Columns (VWR PI89889) were used to desalt the protein to remove loosely associated TPP. Column preparation was performed according to the manufacturer's instructions. All centrifugation steps were conducted at 1000×g for 2 minutes. The first centrifugation step removed the manufacturer's storage buffer. Four wash steps, consisting of the addition of 2 mL 100 mM HEPES, pH 6.8 to the column and followed by centrifugation, prepared the column for desalting the cell lysate. After the wash steps were finished, the column was placed in a fresh 15 mL Corning tube (VWR, 21008-670) and 600 μL of the cell lysate was added to the desalting column and centrifuged. The effluent was collected and analyzed for TPP dependent KivD activity.

KIVD Enzyme Assay Protocol—Coupled

The rates for the conversion of α-ketoisovalerate to isobutryaldehyde were measured in a horse liver ADH (hADH) coupled enzyme assay, which has been described in Gocke, D. et al. (Adv. Synth. Catal. 2007, 349, 1425-1435). Assays of cell free extracts were performed at 30° C. in buffer containing 100 mM HEPES pH 6.8, 10 mM MgCl₂, 200 μM NADH, 30 mM α-KIV (Sigma), and 0.45 U hADH (1 mg=1.5 U) (Sigma). TPP concentration was varied. The oxidation of NADH was monitored at 340 nm in a 1 cm path length cuvette on a Cary 300 spectrophotometer (Agilent Technologies, Wilmington Del.) The enzyme rate was calculated using the molar extinction coefficient of 6220 M⁻¹ cm⁻¹ for NADH. Controls at various concentrations of hADH and cell free extract ensured that measured rate was determined by KIVD enzyme activity.

The specific activities of the putative KIVD enzymes expressed in E. coli were calculated from measured enzyme activities and total protein concentrations. These specific activities were plotted against TPP concentration. Using Kaleidagraph (Synergy), the resulting curves were fit to the saturation equation (SA=(SA_(max)*[TPP])/(K_(c)+[TPP]))+C). The TPP cofactor activation constants (K_(c)) were determined and values are listed in Table 10.

TABLE 10 TPP Cofactor Activation Constants for KivD Putatives Activation ID Constant K_(c), μM KivD80 50 kivD81 0.79 Mca 0.53 KdcA 0.81 KivD 73.4

Example 5 KIVD Activity of Selected Round 2 Enzymes Following Expression in E. coli with Thiamine Supplemented Media

Based on sequence homology to enzymes that possess KIVD activity, five of the round 2 candidates were analyzed further. Vector without an insert and Mca were employed as negative and positive controls, respectively. Expression in E. coli was performed as described in Example 2, with the modification that 30 mg/L thiamine hydrochloride (Sigma) was supplemented in the growth media. Crude extracts were prepared and assayed as described in Example 3. SDS-PAGE analysis indicated that Npu was expressed in E. coli as an insoluble protein.

TABLE 11 KIVD activities for enzymes expressed with thiamine supplemented media Specific Activity ID (U/mg) Npu <0.1 Bme 2.17 Sve 0.50 Hmu 1.25 Cac 0.81 Mca 73.7 Vector control <0.1

Example 6 Measurement of Substrate Specificity Ratios

Two putative KIVD enzymes (KivD81, Mca), KdcA and Lactis KivD, were evaluated for substrate preference. Plasmids (see examples 1 and 2 for plasmid description) encoding for each of the enzymes were isolated and transformed into electrocompetent KEIO Aldh strain of E. coli (Open Biosystems, Huntsville, Ala.). Following shake flask growth at 30° C. to OD600 nm=0.5 in LB media supplemented with 100 ug ampicillin/mL, 1 mM IPTG was added and the cultures grown for an additional 14-16 hrs. Crude cell lysates were generated as follows and the putative KivD enzymes were analyzed in regard to activity with aKiv and pyruvate. Preparation of Cell Free Extract

E. coli cells were suspended in 3 mL of 100 mM HEPES, pH 6.8, 10 mM MgCl₂ and broken by sonication at 0° C. The cells were sonicated with a probe for 10 seconds, with 25 seconds of rest. This cycle was repeated 12 times, for a total of 2 minutes of sonication. The crude extract from the broken cells was centrifuged to pellet the cell debris. The supernatants were removed and stored on ice until assayed.

Protein Quantification

The total protein concentration in cell free extracts was measured by the Bradford Assay using Coomassie Plus (Thermo Scientific #23238, Rockford, Ill.). BSA was employed as a standard. The concentration of protein was measured by following absorbance at 595 nm using a Cary 50 spectrophotometer (Agilent Technologies, Wilmington Del.). KIVD Enzyme Assay Protocol—Coupled The rates for the conversion of ketoisovalerate to isobutryaldehyde were measured in a horse liver ADH (hADH) coupled enzyme assay, which has been described in Gocke, D. et al. (Adv. Synth. Catal. 2007, 349, 1425-1435). Assays of cell free extracts were performed at 30° C. in buffer containing 100 mM HEPES pH 6.8, 10 mM MgCl₂, 200 μM NADH, 500 μM TPP, and 0.45 U hADH (1 mg=1.5 U) (Sigma). The substrate, pyruvate (Sigma) or αKIV (Sigma) was added at various concentrations. The oxidation of NADH was monitored at 340 nm in a 1 cm path length cuvette on a Cary 300 spectrophotometer (Agilent Technologies, Wilmington Del.) The enzyme rate was calculated using the molar extinction coefficient of 6220 M⁻¹ cm⁻¹ for NADH. Controls at various concentrations of hADH and cell free extract ensured that measured rate was determined by KIVD enzyme activity.

The specific activities of the putative KIVD enzymes expressed in E. coli were calculated from measured enzyme activities and total protein concentrations. These specific activities were plotted against the appropriate substrate. Using Kaleidagraph (Synergy), the resulting curves were fit to the Michaelis-Menton equation and K_(m) and V_(max) were determined. Kinetic values and specificity ratio ([(V_(max)/K_(m))αKiv]/[(V_(max)/K_(m))pyruvate]) are listed in Table 12.

TABLE 12 Kinetic Values of KivD Putatives a-KIV pyruvate Km, Vmax, Vmax/Km, Km, Vmax, Vmax/Km, Specificity Ratio ID mM U/mg ml/(min * mg) mM U/mg ml/(min * mg) (α-Kiv/Pyruvate) Mca 0.7 4.0 5.6 4.3 0.1 0.03 193.7 Lactis 2.5 43.2 17.5 16.5 0.9 0.05 320.9 KdcA 2.2 24.5 11.2 16.0 0.5 0.03 356.6 KivD 81 1.4 5.5 3.9 15.7 0.2 0.02 255.3

Example 7 Construction of PNY1503(BP1064)

Construction of Saccharomyces cerevisiae Strain BP1064 (PNY1503)

The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6, and GPD2.

Deletions, which completely removed the entire coding sequence, were created by homologous recombination with PCR fragments containing regions of homology upstream and downstream of the target gene and either a G418 resistance marker or URA3 gene for selection of transformants. The G418 resistance marker, flanked by loxP sites, was removed using Cre recombinase (pRS423::PGAL1-cre; SEQ ID NO: 71). The URA3 gene was removed by homologous recombination to create a scarless deletion, or if flanked by loxP sites was removed using Cre recombinase.

The scarless deletion procedure was adapted from Akada et al., Yeast, 23:399, 2006. In general, the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene). Fragments A and C corresponded to the 500 bp immediately upstream of the target gene (Fragment A) and the 500 bp of the 3′ end of the target gene (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target gene and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome. Using the PCR product ABUC cassette, the URA3 marker was first integrated into and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in the PCR cassette between fragments A and B.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxP cassette was PCR-amplified from pLA54 template DNA (SEQ ID NO:72). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP sites to allow recombination with Cre recombinase and removal of the marker. PCR was done using Phusion DNA polymerase and primers BK505 and BK506 (SEQ ID NOs:73 and 74). The URA3 portion of each primer was derived from the 5′ region upstream of the URA3 promoter and 3′ region downstream of the coding region such that integration of the loxP-kanMX-loxP marker resulted in replacement of the URA3 coding region. The PCR product was transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YPD containing G418 (100 μg/ml) at 30° C. Transformants were screened to verify correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs:75 and 76) and designated CEN.PK 113-7D Δura3::kanMX.

HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 77) and primer oBP453 (SEQ ID NO: 78), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO: 79), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 80), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 81), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 82), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 83), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 84). PCR products were purified with a PCR Purification kit (Qiagen). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 77) and oBP455 (SEQ ID NO: 80). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO: 81) and oBP459 (SEQ ID NO: 84). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 77) and oBP459 (SEQ ID NO: 84). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a his3 knockout were screened for by PCR with primers oBP460 (SEQ ID NO: 85) and oBP461 (SEQ ID NO: 86) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was selected as strain CEN.PK 113-7D Δura3::kanMX Δhis3::URA3.

KanMX Marker Removal from the Dura3 Site and URA3 Marker Removal from the Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMX Δhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 71) using a Frozen-EZ Yeast Transformation II kit (Zymo Research) and plating on synthetic complete medium lacking histidine and uracil supplemented with 2% glucose at 30° C. Transformants were grown in YP supplemented with 1% galactose at 30° C. for ˜6 hours to induce the Cre recombinase and KanMX marker excision and plated onto YPD (2% glucose) plates at 30° C. for recovery. An isolate was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in and plated on YPD for removal of the pRS423::PGAL1-cre plasmid. Isolates were checked for loss of the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by assaying growth on YPD+G418 plates, synthetic complete medium lacking uracil plates, and synthetic complete medium lacking histidine plates. A correct isolate that was sensitive to G418 and auxotrophic for uracil and histidine was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletions and marker removal were confirmed by PCR and sequencing with primers oBP450 (SEQ ID NO: 87) and oBP451 (SEQ ID NO: 88) for Δura3 and primers oBP460 (SEQ ID NO: 85) and oBP461 (SEQ ID NO: 86) for Δhis3 using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen).

PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO: 89) and primer oBP441 (SEQ ID NO: 90), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B. PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 91), containing a 5′ tail with homology to the 3″ end of PDC6 Fragment A, and primer oBP443 (SEQ ID NO: 92), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primer oBP444 (SEQ ID NO: 93), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 94), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C was amplified with primer oBP446 (SEQ ID NO: 95), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO: 96). PCR products were purified with a PCR Purification kit (Qiagen). PDC6 Fragment AB was created by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment B and amplifying with primers oBP440 (SEQ ID NO: 89) and oBP443 (SEQ ID NO: 92). PDC6 Fragment UC was created by overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C and amplifying with primers oBP444 (SEQ ID NO: 93) and oBP447 (SEQ ID NO: 96). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC6 ABUC cassette was created by overlapping PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying with primers oBP440 (SEQ ID NO: 89) and oBP447 (SEQ ID NO: 96). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made and transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc6 knockout were screened for by PCR with primers oBP448 (SEQ ID NO: 97) and oBP449 (SEQ ID NO: 98) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 97) and oBP449 (SEQ ID NO: 98) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC6 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 99) and oBP555 (SEQ ID NO: 100). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designated as BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC #700610. The A fragment followed by the ilvD coding region from Streptococcus mutans for the PCR cassette for the PDC1 deletion-ilvDSm integration was amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and NYLA83 (described in U.S. App. Pub. No. 20110124060, incorporated herein by reference in its entirety) genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). (NYLA83 is a strain that carries the PDC1 deletion ilvDSm integration described in U.S. Patent Application Publication No. 2009/0305363, which is herein incorporated by reference in its entirety.) PDC1 Fragment A-ilvDSm (SEQ ID NO: 101) was amplified with primer oBP513 (SEQ ID NO: 102) and primer oBP515 (SEQ ID NO: 103), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment B. The B, U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSm integration were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 Fragment B was amplified with primer oBP516 (SEQ ID NO: 104) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 105), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO: 106), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B, and primer oBP519 (SEQ ID NO: 107), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primer oBP520 (SEQ ID NO: 108), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 109). PCR products were purified with a PCR Purification kit (Qiagen). PDC1 Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513 (SEQ ID NO: 102) and oBP517 (SEQ ID NO: 105). PDC1 Fragment UC was created by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and amplifying with primers oBP518 (SEQ ID NO: 106) and oBP521 (SEQ ID NO: 109). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC1 A-ilvDSm-BUC cassette was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying with primers oBP513 (SEQ ID NO: 102) and oBP521 (SEQ ID NO: 109). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc1 knockout ilvDSm integration were screened for by PCR with primers oBP511 (SEQ ID NO: 110) and oBP512 (SEQ ID NO: 111) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC1 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO:112) and oBP551 (SEQ ID NO:113). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC1, integration of ilvDSm, and marker removal were confirmed by PCR and sequencing with primers oBP511 (SEQ ID NO:110) and oBP512 (SEQ ID NO:111) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm and designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region from Achromobacter xylosoxidans (The sadB gene is described in U.S. Patent Appl. Pub. No. 2009/0269823, which is herein incorporated by reference in its entirety). A segment of the PCR cassette for the PDC5 deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.

pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 gene from Saccharomyces cerevisiae situated within a multiple cloning site (MCS). pUC19 contains the pMB1 replicon and a gene coding for beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the sequences from upstream and downstream of this gene were included for expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.

The DNA encompassing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region from Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438 (SEQ ID NO: 114), containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 115), containing XbaI, PacI, and NotI restriction sites, using Phusion High-Fidelity PCR Master Mix (New England BioLabs). Genomic DNA was prepared using a Gentra Puregene Yeast/Bact kit (Qiagen). The PCR product and pUC19 (SEQ ID NO: 116) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to create vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO: 117) and oBP265 (SEQ ID NO: 118).

The coding sequence of sadB and PDC5 Fragment B were cloned into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. The coding sequence of sadB was amplified using pLH468-sadB (SEQ ID NO: 119) as template with primer oBP530 (SEQ ID NO: 120), containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 121), containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B. PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 122), containing a 5′ tail with homology to the 3′ end of sadB, and primer oBP533 (SEQ ID NO:123), containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). sadB-PDC5 Fragment B was created by overlapping PCR by mixing the sadB and PDC5 Fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 120) and oBP533 (SEQ ID NO: 123). The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. The resulting plasmid was used as a template for amplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 124) and oBP546 (SEQ ID NO: 125), containing a 5′ tail with homology to the 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547 (SEQ ID NO: 126) containing a 5′ tail with homology to the 3′ end of PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 127). PCR products were purified with a PCR Purification kit (Qiagen). PDC5 sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C and amplifying with primers oBP536 (SEQ ID NO: 124) and oBP539 (SEQ ID NO: 127). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 128) was created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ ID NO: 129), containing a 5′ tail with homology to the 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 127). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose) at 30° C. Transformants with a pdc5 knockout sadB integration were screened for by PCR with primers oBP540 (SEQ ID NO: 130) and oBP541 (SEQ ID NO: 131) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC5 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 132) and oBP553 (SEQ ID NO: 133). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3.

CEN.PK 113-7D &ura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 was grown overnight in YPE (1% ethanol) and plated on synthetic complete medium supplemented with ethanol (no glucose) and containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC5, integration of sadB, and marker removal were confirmed by PCR with primers oBP540 (SEQ ID NO: 130) and oBP541 (SEQ ID NO: 131) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB and designated as BP913.

GPD2 Deletion

To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxP cassette (SEQ ID NO: 134) was PCR-amplified using loxP-URA3-loxP PCR (SEQ ID NO: 135) as template DNA. loxP-URA3-loxP contains the URA3 marker from (ATCC #77107) flanked by loxP recombinase sites. PCR was done using Phusion DNA polymerase and primers LA512 and LA513 (SEQ ID NOs: 136 and 137). The GPD2 portion of each primer was derived from the 5′ region upstream of the GPD2 coding region and 3′ region downstream of the coding region such that integration of the loxP-URA3-loxP marker resulted in replacement of the GPD2 coding region. The PCR product was transformed into BP913 and transformants were selected on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose). Transformants were screened to verify correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs:138 and 139).

The URA3 marker was recycled by transformation with pRS423::PGAL1-cre (SEQ ID NO: 71) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30 C. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and incubated at 30 C to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid. The deletion and marker removal were confirmed by PCR with primers oBP582 (SEQ ID NO: 138) and oBP591 (SEQ ID NO: 140). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as BP1064 (PNY1503).

Example 8 Construction of PNY1507

Construction of Saccharomyces cerevisiae Strains BP1135 (PNY1505) and PNY1507 and Isobutanol-Producing Derivatives

The purpose of this Example was to construct Saccharomyces cerevisiae strains BP1135 and PNY1507. These strains were derived from PNY1503 (BP1064). The construction of PNY1503 (BP1064) is described above. BP1135 contains an additional deletion of the FRA2 gene. PNY1507 was derived from BP1135 with additional deletion of the ADH1 gene, with integration of the kivD gene from Lactococcus lactis, codon optimized for expression in Saccharomyces cerevisiae, into the ADH1 locus.

FRA2 Deletion

The FRA2 deletion was designed to delete 250 nucleotides from the 3′ end of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding sequence intact. An in-frame stop codon was present 7 nucleotides downstream of the deletion. The four fragments for the PCR cassette for the scarless FRA2 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). FRA2 Fragment A was amplified with primer oBP594 (SEQ ID NO: 141) and primer oBP595 (SEQ ID NO: 142), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment B. FRA2 Fragment B was amplified with primer oBP596 (SEQ ID NO: 143), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment A, and primer oBP597 (SEQ ID NO: 144), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment U. FRA2 Fragment U was amplified with primer oBP598 (SEQ ID NO: 145), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO: 146), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment C. FRA2 Fragment C was amplified with primer oBP600 (SEQ ID NO: 147), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment U, and primer oBP601 (SEQ ID NO: 148). PCR products were purified with a PCR Purification kit (Qiagen). FRA2 Fragment AB was created by overlapping PCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying with primers oBP594 (SEQ ID NO: 141) and oBP597 (SEQ ID NO: 144). FRA2 Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U and FRA2 Fragment C and amplifying with primers oBP598 (SEQ ID NO: 145) and oBP601 (SEQ ID NO: 148). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The FRA2 ABUC cassette was created by overlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO: 141) and oBP601 (SEQ ID NO: 148). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of PNY1503 were made and transformed with the FRA2 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Transformants with a fra2 knockout were screened for by PCR with primers oBP602 (SEQ ID NO:149) and oBP603 (SEQ ID NO: 150) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was grown in YPE (yeast extract, peptone, 1% ethanol) and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion and marker removal were confirmed by PCR with primers oBP602 (SEQ ID NO: 149) and oBP603 (SEQ ID NO: 150) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the FRA2 gene from the isolate was demonstrated by a negative PCR result using primers specific for the deleted coding sequence of FRA2, oBP605 (SEQ ID NO: 151) and oBP606 (SEQ ID NO: 152). The correct isolate was selected as strain CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ and designated as PNY1505 (BP1135). This strain was transformed with isobutanol pathway plasmids (pYZ090, SEQ ID NO: 69) and pLH468 (SEQ ID NO: 70), and one clone was designated BP1168 (PNY1506).

ADH1 Deletion and kivD LI(y) Integration

The ADH1 gene was deleted and replaced with the kivD coding region from Lactococcus lactis codon optimized for expression in Saccharomyces cerevisiae. The scarless cassette for the ADH1 deletion-kivD_LI(y) integration was first cloned into plasmid pUC19-URA3MCS.

The kivD coding region from Lactococcus lactis codon optimized for expression in Saccharomyces cerevisiae was amplified using pLH468 (SEQ ID NO: 70) as template with primer oBP562 (SEQ ID NO: 153), containing a PmeI restriction site, and primer oBP563 (SEQ ID NO: 154), containing a 5′ tail with homology to the 5′ end of ADH1 Fragment B. ADH1 Fragment B was amplified from genomic DNA prepared as above with primer oBP564 (SEQ ID NO: 155), containing a 5′ tail with homology to the 3′ end of kivD_LI(y), and primer oBP565 (SEQ ID NO: 156), containing a FseI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). kivD_LI(y)-ADH1 Fragment B was created by overlapping PCR by mixing the kivD_LI(y) and ADH1 Fragment B PCR products and amplifying with primers oBP562 (SEQ ID NO: 153) and oBP565 (SEQ ID NO: 156). The resulting PCR product was digested with PmeI and FseI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. ADH1 Fragment A was amplified from genomic DNA with primer oBP505 (SEQ ID NO: 157), containing a SacI restriction site, and primer oBP506 (SEQ ID NO: 158), containing an AscI restriction site. The ADH1 Fragment A PCR product was digested with SacI and AscI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1 Fragment B. ADH1 Fragment C was amplified from genomic DNA with primer oBP507 (SEQ ID NO: 159), containing a PacI restriction site, and primer oBP508 (SEQ ID NO: 160), containing a SalI restriction site. The ADH1 Fragment C PCR product was digested with PacI and SalI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B. The hybrid promoter UAS(PGK1)-P_(FBA1) was amplified from vector pRS316-UAS(PGK1)-P_(FBA1)-GUS (SEQ ID NO: 161) with primer oBP674 (SEQ ID NO: 162), containing an AscI restriction site, and primer oBP675 (SEQ ID NO: 163), containing a PmeI restriction site. The UAS(PGK1)-P_(FBA1) PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1 Fragments ABC. The entire integration cassette was amplified from the resulting plasmid with primers oBP505 (SEQ ID NO: 157) and oBP508 (SEQ ID NO: 160) and purified with a PCR Purification kit (Qiagen).

Competent cells of PNY1505 were made and transformed with the ADH1-kivD_LI(y) PCR cassette constructed above using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of ADH1 and integration of kivD_LI(y) were confirmed by PCR with external primers oBP495 (SEQ ID NO: 164) and oBP496 (SEQ ID NO: 165) and with kivD_LI(y) specific primer oBP562 (SEQ ID NO: 153) and external primer oBP496 (SEQ ID NO: 165) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ adh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1t and designated as PNY1507 (BP1201).

Example 9 Construction of PNY2211

Construction of S. cerevisiae Strain PNY2211 and PNY2209

PNY2211 was constructed in several steps from S. cerevisiae strain PNY1507 as described in the following paragraphs. First the strain was modified to contain a phosophoketolase gene. Next, an acetolactate synthase gene (alsS) was added to the strain, using an integration vector targeted to sequence adjacent to the phosphoketolase gene. Finally, homologous recombination was used to remove the phosphoketolase gene and integration vector sequences, resulting in a scarless insertion of alsS in the intergenic region between pdc1Δ::ilvD and the native TRX1 gene of chromosome XII. The resulting genotype of PNY2211 is MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH| sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ adh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1t.

A phosphoketolase gene cassette was introduced into PNY1507 by homologous recombination. The integration construct was generated as follows. The plasmid pRS423::CUP1-alsS+FBA-budA (previously described in US2009/0305363, which is herein incorporated by reference in its entirety) was digested with NotI and XmaI to remove the 1.8 kb FBA-budA sequence, and the vector was religated after treatment with Klenow fragment. Next, the CUP1 promoter was replaced with a TEF1 promoter variant (M4 variant previously described by Nevoigt et al. Appl. Environ. Microbiol. 72: 5266-5273 (2006), which is herein incorporated by reference in its entirety) via DNA synthesis and vector construction service from DNA2.0 (Menlo Park, Calif.). The resulting plasmid, pRS423::TEF(M4)-alsS was cut with StuI and MluI (removes 1.6 kb portion containing part of the alsS gene and CYC1 terminator), combined with the 4 kb PCR product generated from pRS426::GPD-xpk1+ADH-eutD (SEQ ID NO: 167) with primers N1176 (SEQ ID NO: 168) and N1177 (SEQ ID NO: 169) and an 0.8 kb PCR product DNA generated from yeast genomic DNA (ENO1 promoter region) with primers N822 (SEQ ID NO: 170) and N1178 (SEQ ID NO: 171) and transformed into S. cerevisiae strain BY4741 (ATCC #201388); gap repair cloning methodology, see Ma et al. Gene 58:201-216 (1987). Transformants were obtained by plating cells on synthetic complete medium without histidine. Proper assembly of the expected plasmid (pRS423::TEF(M4)-xpk1+ENO1-eutD, SEQ ID NO: 172) was confirmed by PCR (primers N821 (SEQ ID NO: 173) and N1115 (SEQ ID NO: 174)) and by restriction digest (BglI). Two clones were subsequently sequenced. The 3.1 kb TEF(M4)-xpk1 gene was isolated by digestion with SacI and NotI and cloned into the pUC19-URA3::ilvD-TRX1 vector (Clone A, cut with AflII). Cloning fragments were treated with Klenow fragment to generate blunt ends for ligation. Ligation reactions were transformed into E. coli StbI3 cells, selecting for ampicillin resistance. Insertion of TEF(M4)-xpk1 was confirmed by PCR (primers N1110 (SEQ ID NO: 175) and N1114 (SEQ ID NO: 176)). The vector was linearized with AflII and treated with Klenow fragment. The 1.8 kb KpnI-HinclI geneticin resistance cassette was cloned by ligation after Klenow fragment treatment. Ligation reactions were transformed into E. coli StbI3 cells, selecting for ampicillin resistance. Insertion of the geneticin cassette was confirmed by PCR (primers N160SeqF5 (SEQ ID NO: 183) and BK468 (SEQ ID NO: 178)). The plasmid sequence is provided as SEQ ID NO: 179 (pUC19-URA3::pdc1::TEF(M4)-xpk1::kan).

The resulting integration cassette (pdc1::TEF(M4)-xpk1::KanMX::TRX1) was isolated (AscI and Nael digestion generated a 5.3 kb band that was gel purified) and transformed into PNY1507 using the Zymo Research Frozen-EZ Yeast Transformation Kit (Cat. No. T2001). Transformants were selected by plating on YPE plus 50 μg/ml G418. Integration at the expected locus was confirmed by PCR (primers N886 (SEQ ID NO: 180) and N1214 (SEQ ID NO: 181)). Next, plasmid pRS423::GAL1p-Cre (SEQ ID NO: 71), encoding Cre recombinase, was used to remove the loxP-flanked KanMX cassette. Proper removal of the cassette was confirmed by PCR (primers oBP512 (SEQ ID NO: 111) and N160SeqF5 (SEQ ID NO: 177)). Finally, the alsS integration plasmid (SEQ ID NO: 225, pUC19-kan::pdc1::FBA-alsS::TRX1, clone A) was transformed into this strain using the included geneticin selection marker. Two integrants were tested for acetolactate synthase activity by transformation with plasmids pYZ090ΔalsS (SEQ ID NO: 182) and pBP915 (SEQ ID NO: 166) (transformed using Protocol #2 in Amberg, Burke and Strathern “Methods in Yeast Genetics” (2005)), and evaluation of growth and isobutanol production in glucose-containing media (methods for growth and isobutanol measurement are as follows: All strains were grown in synthetic complete medium, minus histidine and uracil containing 0.3% glucose and 0.3% ethanol as carbon sources (10 mL medium in 125 mL vented Erlenmeyer flasks (VWR Cat. No. 89095-260). After overnight incubation (30° C., 250 rpm in an Innova®40 New Brunswick Scientific Shaker), cultures were diluted back to 0.2 OD (Eppendorf BioPhotometer measurement) in synthetic complete medium containing 2% glucose and 0.05% ethanol (20 ml medium in 125 mL tightly-capped Erlenmeyer flasks (VWR Cat. No. 89095-260)). After 48 hours incubation (30° C., 250 rpm in an Innova®40 New Brunswick Scientific Shaker), culture supernatants (collected using Spin-X centrifuge tube filter units, Costar Cat. No. 8169) were analyzed by HPLC per methods described in U.S. Appl. Pub. No. 20070092957).). One of the two clones was positive and was named PNY2218.

PNY2218 was treated with Cre recombinase, and the resulting clones were screened for loss of the xpk1 gene and pUC19 integration vector sequences by PCR (primers N886 (SEQ ID NO: 180) and N160SeqR5 (SEQ ID NO: 183)). This left only the alsS gene integrated in the pdc1-TRX1 intergenic region after recombination of the DNA upstream of xpk1 and the homologous DNA introduced during insertion of the integration vector (a “scarless” insertion since vector, marker gene and loxP sequences are lost). Although this recombination could have occurred at any point, the vector integration appeared to be stable even without geneticin selection, and the recombination event was only observed after introduction of the Cre recombinase. One clone was designated PNY2211.

Example 10 Construction of PNY1528

PNY1528 (hADH Integrations in PNY2211)

Deletions/integrations were created by homologous recombination with PCR products containing regions of homology upstream and downstream of the target region and the URA3 gene for selection of transformants. The URA3 gene was removed by homologous recombination to create a scarless deletion/integration.

The scarless deletion/integration procedure was adapted from Akada et al., Yeast, 23:399 (2006). The PCR cassette for each deletion/integration was made by combining four fragments, A-B-U-C, and the gene to be integrated by cloning the individual fragments into a plasmid prior to the entire cassette being amplified by PCR for the deletion/integration procedure. The gene to be integrated was included in the cassette between fragments A and B. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene) regions. Fragments A and C (each approximately 100 to 500 bp long) corresponded to the sequence immediately upstream of the target region (Fragment A) and the 3′ sequence of the target region (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target region and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome.

YPRCΔ15 Deletion and Horse Liver Adh Integration

The YPRCΔ15 locus was deleted and replaced with the horse liver adh gene, codon optimized for expression in Saccharomyces cerevisiae, along with the PDC5 promoter region (538 bp) from Saccharomyces cerevisiae and the ADH1 terminator region (316 bp) from Saccharomyces cerevisiae. The scarless cassette for the YPRCΔ15 deletion-P[PDC5]-adh_HL(y)-ADH1t integration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). YPRCΔ15 Fragment A was amplified from genomic DNA with primer oBP622 (SEQ ID NO: 184), containing a KpnI restriction site, and primer oBP623 (SEQ ID NO: 185), containing a 5′ tail with homology to the 5′ end of YPRCΔ15 Fragment B. YPRCΔ15 Fragment B was amplified from genomic DNA with primer oBP624 (SEQ ID NO: 186), containing a 5′ tail with homology to the 3′ end of YPRCΔ15 Fragment A, and primer oBP625 (SEQ ID NO: 187), containing a FseI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). YPRCΔ15 Fragment A—YPRCΔ15 Fragment B was created by overlapping PCR by mixing the YPRCΔ15 Fragment A and YPRCΔ15 Fragment B PCR products and amplifying with primers oBP622 (SEQ ID NO: 184) and oBP625 (SEQ ID NO: 187). The resulting PCR product was digested with KpnI and FseI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. YPRCΔ15 Fragment C was amplified from genomic DNA with primer oBP626 (SEQ ID NO: 188), containing a NotI restriction site, and primer oBP627 (SEQ ID NO: 189), containing a PacI restriction site. The YPRCΔ15 Fragment C PCR product was digested with NotI and PacI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing YPRCΔ15 Fragments AB. The PDC5 promoter region was amplified from CEN.PK 113-7D genomic DNA with primer HY21 (SEQ ID NO: 190), containing an AscI restriction site, and primer HY24 (SEQ ID NO: 191), containing a 5′ tail with homology to the 5′ end of adh_HI(y). adh_HI(y)-ADH1t was amplified from pBP915 (SEQ ID NO: 166) with primers HY25 (SEQ ID NO: 192), containing a 5′ tail with homology to the 3′ end of P[PDC5], and HY4 (SEQ ID NO: 193), containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). P[PDC5]-adh_HL(y)-ADH1t was created by overlapping PCR by mixing the P[PDC5] and adh_HL(y)-ADH1t PCR products and amplifying with primers HY21 (SEQ ID NO: 190) and HY4 (SEQ ID NO: 193). The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing YPRCΔ15 Fragments ABC. The entire integration cassette was amplified from the resulting plasmid with primers oBP622 (SEQ ID NO: 184) and oBP627 (SEQ ID NO: 189).

Competent cells of PNY2211 were made and transformed with the YPRCΔ15 deletion-P[PDC5]-adh_HL(y)-ADH1t integration cassette PCR product using a Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30 C. Transformants were screened for by PCR with primers URA3-end F (SEQ ID NO: 194) and oBP637 (SEQ ID NO: 195). Correct transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium supplemented with 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion of YPRCΔ15 and integration of P[PDC5]-adh_HL(y)-ADH1t were confirmed by PCR with external primers oBP636 (SEQ ID NO: 124) and oBP637 (SEQ ID NO: 195) using genomic DNA prepared with a YeaStar Genomic DNA kit (Zymo Research). A correct isolate of the following genotype was selected for further modification: CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ adh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)ADH it yprcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t.

Horse Liver Adh Integration at Fra2

The horse liver adh gene, codon optimized for expression in Saccharomyces cerevisiae, along with the PDC1 promoter region (870 bp) from Saccharomyces cerevisiae and the ADH1 terminator region (316 bp) from Saccharomyces cerevisiae, was integrated into the site of the fra2 deletion. The scarless cassette for the fra2Δ-P[PDC1]-adh_HL(y)-ADH1t integration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). fra2Δ Fragment C was amplified from genomic DNA with primer oBP695 (SEQ ID NO: 197), containing a NotI restriction site, and primer oBP696 (SEQ ID NO: 198), containing a PacI restriction site. The fra2Δ Fragment C PCR product was digested with NotI and PacI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS. fra2Δ Fragment B was amplified from genomic DNA with primer oBP693 (SEQ ID NO: 199), containing a PmeI restriction site, and primer oBP694 (SEQ ID NO: 200), containing a FseI restriction site. The resulting PCR product was digested with PmeI and FseI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing fra2Δ fragment C after digestion with the appropriate enzymes. fra2Δ Fragment A was amplified from genomic DNA with primer oBP691 (SEQ ID NO: 201), containing BamHI and AsiSI restriction sites, and primer oBP692 (SEQ ID NO: 202), containing AscI and SwaI restriction sites. The fra2Δ fragment A PCR product was digested with BamHI and AscI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing fra2Δ fragments BC after digestion with the appropriate enzymes. The PDC1 promoter region was amplified from CEN.PK 113-7D genomic DNA with primer HY16 (SEQ ID NO: 203), containing an AscI restriction site, and primer HY19 (SEQ ID NO: 204), containing a 5′ tail with homology to the 5′ end of adh_HI(y). adh_HI(y)-ADH1t was amplified from pBP915 with primers HY20 (SEQ ID NO: 205), containing a 5′ tail with homology to the 3′ end of P[PDC1], and HY4 (SEQ ID NO: 193), containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). P[PDC1]-adh_HL(y)-ADH1t was created by overlapping PCR by mixing the P[PDC1] and adh_HL(y)ADH1t PCR products and amplifying with primers HY16 (SEQ ID NO: 203) and HY4 (SEQ ID NO: 193).The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing fra2Δ Fragments ABC. The entire integration cassette was amplified from the resulting plasmid with primers oBP691 (SEQ ID NO: 201) and oBP696 (SEQ ID NO: 198).

Competent cells of the PNY2211 variant with adh_HI(y) integrated at YPRCΔ15 were made and transformed with the fra2Δ-P[PDC1]-adh_HL(y)-ADH1t integration cassette PCR product using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30 C. Transformants were screened for by PCR with primers URA3-end F (SEQ ID NO: 194) and oBP731 (SEQ ID NO: 206). Correct transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium supplemented with 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The integration of P[PDC1]-adh_HL(y)-ADH1t was confirmed by colony PCR with internal primer HY31 (SEQ ID NO: 207) and external primer oBP731 (SEQ ID NO: 155) and PCR with external primers oBP730 (SEQ ID NO: 208) and oBP731 (SEQ ID NO: 206) using genomic DNA prepared with a YeaStar Genomic DNA kit (Zymo Research). A correct isolate of the following genotype was designated PNY1528: CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)ADH1t yprcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t.

Example 11 Construction of Strains PNY1549, PNY1550, and PNY1551

The purpose of this example is to describe the assembly of the constructs used to replace the chromosomal copy of kivD_LI(y) in PNY1528 at the adh1Δ locus with kivD_Lg(y) or kivD_Mc(y) and construction of isobutanologen strains PNY1549, PNY1550, and PNY1551 expressing the kivD genes.

Deletions/integrations were created by homologous recombination with PCR products containing regions of homology upstream and downstream of the target region and the URA3 gene for selection of transformants. The URA3 gene was removed by homologous recombination to create a scarless deletion/integration. The scarless deletion/integration procedure was adapted from Akada et al., Yeast, 23:399, 2006. The PCR cassette for each deletion/integration was made by combining four fragments, A-B-U-C, and the gene to be integrated by cloning the individual fragments into a plasmid prior to the entire cassette being amplified by PCR for the deletion/integration procedure. The gene to be integrated was included in the cassette between fragments A and B. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene) regions. Fragments A and C (500 bp long) corresponded to the sequence immediately upstream of the target region (Fragment A) and the 3′ sequence of the target region (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target region and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome.

The plasmids to integrate kivD_Lg(y) and kivD_Mc(y) were derived from a plasmid constructed to integrate UAS(PGK1)P[FBA1]-kivD_LI(y) into the ADH1 locus of Saccharomyces cerevisiae. Construction of the plasmid used to integrate UAS(PGK1)P[FBA1]-kivD_LI(y) into the ADH1 locus is described below. The plasmids were constructed in pUC19-URA3MCS.

Construction of the ADH1 Deletion/UAS(PGK1)P[FBA1]-kivD_LI(y) Integration Plasmid

The kivD coding region from Lactococcus lactis codon optimized for expression in Saccharomyces cerevisiae, kivD_LI(y), was amplified using pLH468 (SEQ ID NO: 70) as template with primer oBP562 (SEQ ID NO: 153), containing a PmeI restriction site, and primer oBP563 (SEQ ID NO: 154), containing a 5′ tail with homology to the 5′ end of ADH1 Fragment B. ADH1 Fragment B was amplified from Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA with primer oBP564 (SEQ ID NO: 155), containing a 5′ tail with homology to the 3′ end of kivD_LI(y), and primer oBP565 (SEQ ID NO: 156), containing a FseI restriction site. PCR products were purified with a PCR Purification kit (Qiagen; Valencia, Calif.). kivD_LI(y)-ADH1 Fragment B was created by overlapping PCR by mixing the kivD_LI(y) and ADH1 Fragment B PCR products and amplifying with primers oBP562 (SEQ ID NO: 153) and oBP565 (SEQ ID NO 156). The resulting PCR product was digested with PmeI and FseI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. ADH1 Fragment A was amplified from genomic DNA with primer oBP505 (SEQ ID NO: 157), containing a SacI restriction site, and primer oBP506 (SEQ ID NO: 158), containing an AscI restriction site. The ADH1 Fragment A PCR product was digested with SacI and AscI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1 Fragment B. ADH1 Fragment C was amplified from genomic DNA with primer oBP507 (SEQ ID NO: 159), containing a PacI restriction site, and primer oBP508 (SEQ ID NO: 160), containing a SalI restriction site. The ADH1 Fragment C PCR product was digested with PacI and SalI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B. The hybrid promoter UAS(PGK1)-P_(FBA1) (SEQ ID NO: 161) was amplified from vector pRS316-UAS(PGK1)-P_(FBA1)-GUS with primer oBP674 (SEQ ID NO: 162), containing an AscI restriction site, and primer oBP675 (SEQ ID NO: 163), containing a PmeI restriction site. The UAS(PGK1)-P_(FBA1) PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1 Fragments ABC to generate pBP1181.

Construction of pBP1716, pBP1719, and pBP2019

kivD_LI(y) was removed from the ADH1 deletion/UAS(PGK1)P[FBA1]-kivD_LI(y) integration plasmid pBP1181. The plasmid was digested with PmeI and FseI and the large DNA fragment was purified on an agarose gel followed by a gel extraction kit (Qiagen). ADH1 fragment B was amplified from pBP1181 with primer oBP821 (SEQ ID NO: 210), containing a PmeI restriction site, and primer oBP484 (SEQ ID NO: 211), containing a FseI restriction site. The ADH1 fragment B PCR product was digested with PmeI and FseI and ligated with T4 DNA ligase into the corresponding sites of the gel purified large DNA fragment. A PCR fragment corresponding to the 3′ 500 bp of kivD_LI(y) was cloned into the resulting vector for the targeted deletion of kivD_LI(y) in PNY1528. The fragment was amplified from pBP1181 with primers oBP822 (SEQ ID NO: 212), containing a NotI restriction site, and oBP823 (SEQ ID NO: 213), containing a PacI restriction site. The fragment was digested with NotI and PacI and ligated with T4 DNA ligase into the corresponding sites downstream of URA3 in the above plasmid with the kivD_LI(y) deletion after digestion with the appropriate restriction enzymes. The resulting plasmid was designated pBP1716.

The kivD coding region from Listeria grayi codon optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 214), kivD_Lg(y), was synthesized by DNA2.0 (Menlo Park, Calif.). kivD_Lg(y) was amplified with primers oBP828 (SEQ ID NO: 215), containing a PmeI restriction site, and oBP829 (SEQ ID NO: 216) containing a PmeI restriction site. The resulting PCR product was digested with PmeI and ligated with T4 DNA ligase into the corresponding site in pBP1716 after digestion with the appropriate enzyme. The orientation of the cloned gene was checked by PCR with primers FBAp-F (SEQ ID NO: 217) and oBP829 (SEQ ID NO: 216). An isolate with kivD_Lg(y) in the correct orientation was designated pBP1719.

The kivD coding region from Macrococcus caseolyticus codon optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 224), kivD_Mc(y), was synthesized by DNA2.0 (Menlo Park, Calif.). kivD_Mc(y) was amplified with primers oBP900 (SEQ ID NO: 226), containing a PmeI restriction site, and oBP901 (SEQ ID NO: 227) containing a PmeI restriction site. The resulting PCR product was digested with PmeI and ligated with T4 DNA ligase into the corresponding site in pBP1716 after digestion with the appropriate enzyme. The orientation of the cloned gene was checked by PCR with primers FBAp-F (SEQ ID NO: 217) and oBP901 (SEQ ID NO: 227). An isolate with kivD_Mc(y) in the correct orientation was designated pBP2019.

Construction of Strains PNY1549, PNY1550, and PNY1551

Strain PNY1528 was transformed with plasmids pLH702 and pYZ067ΔkivDΔhADH. A transformant was designated PNY1549.

The kivD_LI(y) deletion/kivD_Lg(y) integration cassette was amplified from pBP1719 with primers oBP505 (SEQ ID NO: 73) and oBP823 (SEQ ID NO: 213). Competent cells of the PNY1528 were made and transformed with the PCR product using a Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30 C. Transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium supplemented with 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion of kivD_LI(y) and integration of kivD_Lg(y) was confirmed by PCR with primers oBP674 (SEQ ID NO: 162) and oBP830 (SEQ ID NO: 220) using genomic DNA prepared with a YeaStar Genomic DNA kit (Zymo Research). A correct isolate contained kivD_Lg(y) at the same locus and expressed from the same promoter as kivD_LI(y) in PNY1528. An isolate was transformed with plasmids pLH702 and pYZ067ΔkivDΔhADH. A transformant was designated PNY1550.

The kivD_LI(y) deletion/kivD_Mc(y) integration cassette was amplified from pBP2019 with primers oBP505 (SEQ ID NO: 157) and oBP823 (SEQ ID NO: 213). Competent cells of the PNY1528 were made and transformed with the PCR product using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol at 30 C. Transformants were screened for by PCR with primers HY51 (SEQ ID NO: 221) and oBP906 (SEQ ID NO: 222). Correct transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium supplemented with 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion of kivD_LI(y) and integration of kivD_Mc(y) was confirmed by PCR with primers HY50 (SEQ ID NO: 223) and HY51 (SEQ ID NO: 221) using genomic DNA prepared with a YeaStar Genomic DNA kit (Zymo Research). A correct isolate contained kivD_Mc(y) at the same locus and expressed from the same promoter as kivD_LI(y) in PNY1528. An isolate was transformed with plasmids pLH702 and pYZ067ΔkivDΔhADH. A transformant was designated PNY1551.

Example 12 KIVD Fermentations

Methods:

Inoculum Preparation

For each strain, a single frozen vial was thawed and transferred to 10 mL seed medium in a 125 mL vented shake flask, and incubated at 30° C. and 300 rpm shaking for overnight growth. Five mL of the overnight culture was then transferred to a 250 mL vented shake flask with 75 mL of the seed medium for overnight growth at 30° C. and 300 rpm shaking. When the culture reached OD600 1-2, the flask culture was used to inoculate the 1 L fermenter. The seed medium composition is as follows: yeast nitrogen base without amino acids (Difco), 6.7 g/L; Yeast Synthetic Drop-out Medium Supplements without histidine, leucine, tryptophan and uracil (Sigma), 2.8 g/L; L-leucine, 20 mg/L; L-tryptophan, 4 mg/L; Thiamine HCl, 20 mg/L; niacin, 20 mg/L; ethanol, 3 g/L; glucose 10 g/L. The pH was adjusted to 5.2 with 20% potassium hydroxide, and the medium filter sterilized through a 0.22μ filter.

Fermenter Preparation and Operation:

Fermentations were carded out in 1 L Biostat B DCU3 fermenters (Sartorius, USA). Off-gas composition was monitored by a Prima DB mass spectrometer (Thermo Electron Corp., USA). To measure the isobutanol and ethanol in the off-gas, the gas was first passed through a 1 liter Shott bottle with 0.5 L water, placed in an ice water bath, before sending to the mass spectrometer. The temperature of the fermenter was maintained at 30° C., and pH controlled at 5.2 with 20% KOH throughout the entire fermentation. Aeration was controlled at 0.2 standard liters per minute, and agitation controlled at 100 rpm. Dissolved oxygen was not controlled, and was non-detectable for most of the fermentation. Samples were drawn and analyzed for optical density at 600 nm and for glucose concentration by a YSI Select Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio). Using this analysis, glucose was maintained in excess (5-20 g/L) by manual additions of a 50% (w/w) solution.

The medium used for the fermentations was prepared as follows: prior to sterilization, 520 mL water with 4.0 g ammonium sulfate, 2.2 g potassium phosphate monobasic, 1.5 g magnesium sulfate heptahydrate, and 0.2 mL Sigma Antifoam 204 was transferred to the fermenter. The fermenters were sterilized at 121° C. for 30 minutes. After cooling to the set point of 30° C., the post sterilization ingredients were added aeseptically via a pump. The post sterilization ingredients are made in 200 mL total volume: 4.8 mL of a trace mineral solution (prepared in 1 L water: 15 g EDTA, 4.5 g zinc sulfate heptahydrate, 0.8 g manganese chloride dehydrate, 0.3 g cobalt chloride hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g disodium molybdenum dehydrate, 4.5 g calcium chloride dihydrate, 3 g iron sulfate heptahydrate, 1 g boric acid, 0.1 g potassium iodide), 0.8 mL of a vitamin mixture (in 1 L water, 50 mg biotin, 1 g Ca-pantothenate, 1 g nicotinic acid, 25 g myo-inositol, 1 g pyridoxol hydrochloride, 0.2 g p-aminobenzoic acid), 16 g glucose, 3 mL ethanol, 12.8 mg L-leucine, 3.2 mg L-tryptophan, 2.2 g Yeast Synthetic Drop-out Medium Supplements without histidine, leucine, tryptophan and uracil (Sigma), thiamine HCl was added in the amounts indicated in Table 1 for a specific fermentation, and deionized water to bring the volume to 200 mL; the solution was filter sterilized prior to addition to the fermenter. Final volume of the fermenter, post inoculation, was 800 mL.

Measurements of glucose, isobutanol, and other fermentation by-products in the culture supernatant were carried out by HPLC, using a Bio-Rad Aminex HPX-87H column (Bio-Rad, USA), with refractive index (RI) and a diode array (210 nm) detectors, with an Agilent 1100 series HPLC (Agilent, USA). Chromatographic separation was achieved using 0.01 N H₂SO₄ as the mobile phase with a flow rate of 0.6 mL/min and a column temperature of 40° C. The water trap was sampled at the end of the fermentation and analyzed by the same HPLC method. The weight of water in the trap was also measured to determine the total amount of isobutanol and ethanol stripped from the fermenter. The glucose concentration in the feed bottle was determined with a Mettler Toledo RE40 Refractometer (Mettler Toledo, USA) at 20° C. Reported yields are based on glucose consumed. The yield of isobutanol includes isobutanol in the fermentation broth and in the water trap at the last sample. FIG. 2 shows the molar yields of isobutanol and α-ketoisovalerate for each strain at each thiamine concentration. FIG. 3 shows the concentration of α-ketoisovalerate over the fermentation time for each strain at each thiamine concentration.

TABLE 13 Experimental design. All fermenters were prepared as described above, with the following thiamine additions, and strains used. Results are given for the end of fermentation sample, at 70-72 hours elapsed fermentation time. KIVD Yield of amino acid Thiamine- α- Yield of KIVD Source SEQ ID HCl ketoisovalerate isobutanol Experiment Strain organism NO: (mg/L) mol/mol) (mol/mol) A PNY1549 Lactococcus 68 0 0.020 0.711 lactis B PNY1549 Lactococcus 68 1 0.018 0.719 lactis C PNY1549 Lactococcus 68 30 0.015 0.744 lactis D PNY1550 Listeria grayi 52 0 0.008 0.725 E PNY1550 Listeria grayi 52 1 0.008 0.761 F PNY1550 Listeria grayi 52 30 0.009 0.761 G PNY1551 Macrococcus 61 0 0.013 0.742 caseolyticus H PNY1551 Macrococcus 61 1 0.012 0.731 caseolyticus

TABLE Z HMMER2.0 [2.2 g] NAME sel kivd LENG 562 ALPH Amino RF no CS no MAP yes COM hmmbuild -n sel_kivd sel_kivd.hmm sel_kivd.aln.fa NSEQ 170 DATE Wed Apr. 25 16:17:47 2012 CKSUM 3974 XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4 NULT −4 −8455 NULE 595 −1558 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45 531 201 384 −1998 −644 HMM A C D E F G H I K L M N P Q R S T V W Y m->m m->i m->d i->m i->i d->m d->d b->m m->e −125 * −3589 1 −660 −1258 −3102 −2504 −1232 −2786 −1646 −191 −2212 380 4107 −1056 57 −1939 −2210 149 299 −683 −1714 −1356 24 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −5 −8611 −9654 −894 −1115 −701 −1378 −125 * 2 −333 −2802 232 188 −9 −2305 −964 −2872 1309 −2817 2554 −74 −783 1796 162 606 −870 −2423 −2986 −2304 25 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −52 −8855 −4910 −894 −1115 −701 −1378 * * 3 −167 −3177 −897 −224 −3496 −1807 −1342 −3245 584 −3192 3472 487 495 −43 −1431 606 1206 −2798 −3362 −2680 26 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9373 −10416 −894 −1115 −514 −1738 * * 4 −1278 −3309 −1010 −634 −357 −406 −343 −1699 1703 −3324 1723 790 529 1399 −263 −392 845 −774 −3494 −2813 27 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 5 −2071 −1909 −4335 −3705 319 −3610 −2474 685 −1209 −1256 1140 −3229 −3660 1133 −3145 −2692 −581 −796 −2361 3989 28 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 6 −2265 1438 −2171 −294 −4000 −3199 −1756 −3694 495 −3614 −2730 −1845 −3276 109 1310 −491 3142 −3283 −3740 −3153 29 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 95 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 7 −3887 −3370 −6589 −6295 −4112 −6441 −6626 2521 −6287 −2845 −2785 −6121 −6182 −6284 −6491 −5847 −3884 3280 −5948 −5390 30 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 8 1958 −3190 −5727 −6081 −5916 3113 −5211 −5746 −5875 −6004 −5029 −4274 −4282 −5298 −5507 271 −3050 −4411 −6133 −6113 31 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 95 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 9 −35 668 2939 441 −3662 −2834 −158 −3413 −1085 −3358 −2433 −264 −2932 1789 −819 −1749 462 −2964 −3527 −2843 32 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 10 −6225 −5134 −6587 −6948 1827 −6472 895 −5108 −6502 −3671 −4508 −5078 −6326 −5219 −5852 −5721 −6073 −5255 −1896 4623 33 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 95 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 11 −5223 −4578 −7577 −7024 −2613 −7388 −6057 687 −6841 3221 −1384 −7188 −6354 −5518 −6276 −6872 −5060 −2798 −4471 −4700 34 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 12 −388 −2357 −4920 −4290 −8 −4157 −3025 −90 −3898 2720 1829 −371 −4150 −3469 −3685 −3253 −2508 −142 −2795 −2502 35 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 95 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 13 −1312 −3326 3471 −1191 −3636 −2857 −1516 −3375 −19 −1137 −652 −483 −2953 211 −264 −1773 −304 −2943 −3512 −2842 36 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 14 439 482 −5365 −4228 −5765 −4150 −3224 −5238 −1903 −5063 −4348 −3769 −4556 −56 3921 −3535 −3562 −4642 −4970 −4904 37 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 15 −4929 −4319 −7396 −6867 −2328 −7246 −6080 112 −6718 3131 −1465 −6963 −6308 −5551 −6263 −6654 −4800 475 −4565 −4762 38 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 16 1907 −3310 −1691 14 −3630 −2817 2053 −3379 934 −3326 −2400 −34 −2910 320 −124 453 139 −441 −3495 183 39 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 17 −56 −3334 628 2641 −3654 −331 −1491 −3405 −1074 −3350 −2424 −817 −2924 2132 −402 −1266 −803 −1684 −3517 −2834 40 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 18 1279 2713 −4407 −3772 −1845 −3612 −2483 1586 −3367 1485 641 −1380 −3662 −2991 −3168 −2406 −1248 668 −2349 −339 41 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 19 −3670 −4955 −1906 −1907 −6132 3635 −139 −6124 −3779 −6037 −5399 204 −4392 −3184 −4528 −2134 −3907 −5347 −6174 −5241 42 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 20 127 888 −6505 −6193 −4072 −6225 −6315 3224 −6149 −2864 −2769 −5955 −6048 −6110 −6308 −5592 −3804 2290 −5770 −5233 43 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 21 −1404 −3317 1934 1446 −3638 −534 514 −3388 708 −3333 −2406 −842 −2910 484 1364 238 397 −2939 −3500 −2817 44 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 22 −3542 −4840 −959 1493 −5356 −3960 4527 −4961 1266 −4736 −3985 −2578 −4184 −2160 621 −3339 −3428 −4588 −4729 −4266 45 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 23 −4614 −4064 −7106 −6593 −949 −6859 −5831 2651 −6423 2041 887 −6551 −6148 −5470 −6075 −6196 −4514 1432 −4557 −4662 46 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 24 −6152 −5102 −6594 −6934 4451 −6466 −2681 −923 −6494 −4175 −4291 −5098 −6314 −5222 −5861 −5720 −6008 −5089 −1926 728 47 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 25 −6817 −6056 −7068 −7452 −7681 3860 −6790 −8639 −7676 −8090 −7933 −7257 −6558 −7506 −7149 −7192 −7136 −8044 −6471 −7683 48 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 26 −3890 −3451 −6562 −6315 −3054 −6092 −6094 −762 −6251 −2744 −2730 −5971 −6030 −6109 −6344 −5501 −3925 3824 −5423 −4771 49 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 27 −1434 −4223 −5963 −6324 −6579 −4430 −5795 −6684 −6416 −6779 −6008 −5195 4270 −6007 −6104 −4012 −4219 −5504 −6773 −6647 50 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 28 −6817 −6056 −7068 −7452 −7681 3860 −6790 −8639 −7676 −8090 −7933 −7257 −6558 −7506 −7149 −7192 −7136 −8044 −6471 −7683 51 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 29 −7011 −6304 4224 −5696 −7592 −5938 −6173 −8539 −6895 −7973 −7843 −5989 −6423 −6412 −6926 −7057 −7168 −8129 −6463 −7354 52 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 30 −6231 −5144 −6594 −6955 3179 −6466 −2671 −5120 −6516 −4420 −4520 −5096 −6329 −5237 −5876 −5736 −6083 −5279 −1917 4241 53 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 31 −3089 −3913 −2711 −3071 −5966 −3634 −3928 −5661 −4075 −5921 −5129 4262 −4340 1432 −4466 −1393 −511 −4824 −6032 −5471 54 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 32 −5317 −4663 −7620 −7063 −2591 −7400 −6033 −2118 −6856 3317 350 −7238 −6360 −5504 −6263 −6915 −5143 −3017 −4443 −4675 55 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 33 1427 −3312 −1690 714 −3632 −653 1142 −3382 −396 −2032 −2401 −1452 −2909 2371 93 886 49 −2933 −3496 −1434 56 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 34 −6164 −5107 −6607 −6943 4406 −6485 −2694 −4807 −6504 −663 −4182 −5113 −6320 −5226 −5869 −5739 −6014 −5084 −1937 772 57 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10590 −894 −1115 −701 −1378 * * 35 −6540 −5651 −7264 −7405 −4412 −6422 −6465 −4083 −7402 3396 −3353 −7378 −6583 −6726 −6858 −7527 −6525 −4898 −5545 −5865 58 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 36 −4365 −6260 3945 1169 −6500 −4023 −3531 −6583 −4015 −6379 −5878 −2648 −4625 −3243 −5050 −3962 −4480 −6011 −6405 −5420 59 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 37 −1877 −3344 1806 −647 −3661 −2840 3577 −3410 −1090 −827 −2435 351 −2939 1585 132 −849 −1816 −2964 −3528 1209 60 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 38 −3888 −3371 −6591 −6296 −4112 −6445 −6628 3014 −6288 −2845 −2784 −6123 −6184 −6285 −6492 −5851 −3885 2938 −5949 −5392 61 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 39 −2036 −2027 −1692 858 −2001 −3476 −311 2907 −2736 −1142 1071 −2830 −3535 −23 −2811 −2520 −920 1393 −2470 −2102 62 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 40 1971 −3329 1578 244 −3649 −1476 620 −3400 −1068 −3345 −2418 192 −2919 −154 −8 1202 −783 −2951 −3512 −2828 63 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 41 −409 −3453 622 −1232 −3765 −2911 4439 −3515 −1201 −3464 −2549 262 −3029 −602 89 569 −1928 −3072 −3637 −773 64 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 42 −1972 −3458 1098 1443 −3775 −1962 −1591 −3530 741 −3742 −2551 712 2774 −1136 −528 −590 −1333 −3078 −3640 −2948 65 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 43 −400 −3315 1794 438 −3636 920 501 −2681 830 −3331 −2404 781 −2909 1025 464 −535 −257 −794 −3498 −2816 66 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 44 −4352 −3807 −6941 −6518 −3113 −6765 −6118 2949 −6417 1584 1690 −6443 −6191 −5726 −6244 −6138 −4293 1471 −4920 −4907 67 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 45 −798 1286 168 2104 −3636 −1010 35 −3386 851 −3331 −2404 −1451 −2909 615 1532 −538 570 −1537 −3498 −2815 68 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 46 −6146 −5110 −6626 −6951 1846 −6478 −2744 −4716 −6512 −1758 −4082 −5152 −6322 −5252 −5883 −5764 −6004 −5036 5977 −802 69 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 47 −3594 −3158 −6187 −5771 −3651 −5815 −5287 2351 −5594 −2650 689 −5470 −5666 −874 −5607 −5074 −1307 3167 −4922 −4493 70 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 48 −5086 −5209 −6442 −6820 −7267 3818 −6361 −7746 −7109 −7562 −7035 −6182 −1070 −6779 −6728 −5360 −5517 −6730 −6416 −7292 71 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 49 −873 4117 −5719 −5958 −5804 −1912 −5112 −5610 −5689 −5869 −4911 3400 −4262 −5173 −5390 −561 826 −4348 −6030 −5975 72 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 50 2569 4099 −5963 −6297 −5860 −3458 −5528 −5665 −5907 −5938 −4972 −4302 −4725 −5336 −5503 −1218 1837 −4368 −6091 −6072 73 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 51 −6525 −6005 −6086 −6469 −7264 −5910 −6392 −8381 −7104 −7895 −7691 4452 −6421 −6848 −6867 −6767 −6826 −7793 −6362 −7082 74 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 52 −7062 −6299 −5429 3971 −7596 −5972 −6218 −8532 −6862 −7964 −7840 −6101 −6450 −6478 −6844 −7139 −7217 −8138 −6457 −7375 75 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 53 −6540 −5651 −7264 −7405 −4412 −6422 −6465 −4083 −7402 3396 −3353 −7378 −6583 −6726 −6858 −7527 −6525 −4898 −5545 −5865 76 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 54 −6525 −6005 −6086 −6469 −7264 −5910 −6392 −8381 −7104 −7895 −7691 4452 −6421 −6848 −6867 −6767 −6823 −7793 −6362 −7082 77 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 55 3618 −3587 −5871 −6231 −6208 −629 −5473 −6122 −6137 −6333 −5418 −4647 −4637 −5610 −5778 −3275 −3495 −4828 −6207 −6366 78 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 56 2729 −3187 −5769 −6118 −5914 1544 −5216 −5742 −5881 −6001 −5025 −4279 −4281 −5305 −5507 1916 −3057 −4407 −6131 −6113 79 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 57 −7113 −5912 −6980 −7350 −3209 −6288 −4498 −6840 −7294 −6152 −6227 −6478 −6594 −6601 −6729 −7077 −7152 −6886 −3797 4945 80 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 58 3583 −3173 −5811 −6117 −5746 −3469 −5158 −5452 −5764 −5769 390 −4277 −4278 −5255 −5410 −2336 −1310 −4290 −6008 −5951 81 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 59 3647 −3308 −5792 −6140 −5960 −3582 −5280 −5762 −5907 −6047 −5117 −4389 −4391 −5382 −5549 −2967 −1522 −4468 −6131 −6139 82 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 60 −7012 −6305 4224 −5697 −7593 −5939 −6174 −8540 −6896 −7973 −7844 −5991 −6424 −6413 −6927 −7058 −7169 −8130 −6464 −7355 83 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 61 −6818 −6056 −7069 −7453 −7681 3860 −6791 −8640 −7677 −8091 −7934 −7257 −6559 −7507 −7150 −7193 −7137 −8045 −6472 −7684 84 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 62 −7113 −5912 −6980 −7350 −3209 −6288 −4498 −6840 −7294 −6152 −6227 −6478 −6594 −6601 −6729 −7077 −7152 −6886 −3797 4945 85 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 63 3488 −3199 −5761 −6113 −5920 589 −5221 −5743 −5879 −6006 −5034 −4289 −4292 −5311 −5507 −2846 −1572 −4416 −6132 −6117 86 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 64 −7254 −6210 −7079 −7064 −7531 −6170 −6202 −8372 −5727 −7773 −7566 −6974 −6572 −6416 4266 −7571 −7309 −8053 −6330 −7278 87 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 65 −2092 3243 −4408 −242 −1875 −3641 −2513 1735 −3379 1156 1769 −3277 −3690 480 −3191 −2726 221 640 −2386 −2043 88 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 66 −1017 −3538 −2023 −1440 −3896 −3082 −1668 −3610 1975 −3534 818 2213 −3163 −648 2437 −209 −1250 −3184 −3669 −3052 89 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 67 −3632 −4304 −3901 −3727 −6082 3463 −3420 −5645 −504 −5431 −4731 −3605 1433 −3068 −953 −3702 −3806 −4993 −5286 −5166 90 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 68 1878 −2573 −5199 −4591 1602 −4478 −3386 1888 −4221 591 2136 −4127 −4446 −3799 −4021 −3589 −2567 533 −3129 −2836 91 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 69 2465 −3188 −5757 −6108 −5915 2455 −5215 −5743 −5880 −6002 −5026 −4278 −4281 −5303 −5508 1322 −3057 −4408 −6132 −6114 92 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 70 3539 836 −5969 −6272 −5670 −3498 −5219 −4716 −5882 −5552 −4751 −4328 −4304 −5332 −5494 −2861 −2400 −435 −6008 −5947 93 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9548 −10591 −894 −1115 −701 −1378 * * 71 −5083 −4473 −7465 −6867 800 −7242 −5870 −194 −6663 2814 2264 −6966 −6252 −5400 −6127 −6607 −4918 560 −4390 −4621 94 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 72 −213 298 −4956 −4361 −2405 −4220 −3179 1353 −3994 2042 −1568 −3875 −4245 −3643 −3823 −1825 −1647 2175 −3022 −2668 95 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 73 −2613 −3215 −5612 −5924 −5633 −3501 −5118 −2402 −5643 −5748 −4895 −4266 −4307 −5202 −5341 −1256 4001 −4262 −5980 −5840 96 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 74 −5076 −5190 −6693 −7054 −7082 −5329 −6374 −7534 −7100 −7413 −6937 −6271 −5965 −6844 −6683 −5360 4103 −6627 6359 −7090 97 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 75 −1822 −315 −6206 −6197 −4127 −5645 −2721 −3887 −5776 −3780 −3584 −4797 −5691 −4857 −5329 −4859 −4691 −1102 −2021 −2562 98 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 76 −6820 −6058 −7070 −7454 −7683 3860 −6795 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 99 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 77 −5460 −5067 −6930 −7153 −5975 −5869 −6541 −3514 −7191 −5258 −5195 −6738 −6319 −7040 −6859 −6252 −5668 3911 −6198 −6486 100 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 78 −6820 −6058 −7070 −7454 −7683 6860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 101 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 79 −4115 −6183 1150 3685 −6340 −3819 −3304 −6363 −3776 −6182 −5653 −2409 −4425 −3004 −4836 −1417 −4232 −5776 −6380 −5215 102 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 80 −6542 −5652 −7265 −7406 −4414 −6423 −6466 −4085 −7403 3396 −3355 −7379 −6584 −6727 −6859 −7529 −6527 −4900 −5547 −5867 103 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 81 −270 −3200 −5676 −6032 −5921 −1624 −5205 −5753 −5862 −6010 −5037 −4271 −4288 −5289 −5504 3620 −3068 −4419 −6135 −6112 104 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 82 3657 −3391 −5891 −6230 −5863 −3674 −5330 −5497 −5964 −5893 −5090 −4486 −4476 −5459 −5604 −3070 −3284 −2572 −6075 −6040 105 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 83 −148 −3026 −5947 −5468 −3299 −5459 −4693 3121 −5229 813 693 −5109 −5348 −4949 −5158 −4660 −1460 1518 −4376 −4004 106 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 84 −2880 −3576 −3507 −3897 −5991 −864 −4514 −5910 −4994 −6071 −5192 4277 −4364 −4386 −5232 −763 −3320 −4688 −6147 −5811 107 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 85 1559 −3594 −5810 −6173 −6213 3451 −5464 −6130 −6126 −6338 −5423 −4637 −4640 −5595 −5777 −3279 −3500 −4835 −6211 −6367 108 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 86 −3812 −3333 −6439 −6052 −3639 −6164 −5755 3517 −5936 −340 273 −5819 −5913 −5703 −5964 −5470 652 1003 −5167 −4802 109 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 87 3609 −3204 −5772 −6123 −5921 −1837 −5225 −5741 −5878 −6007 −5037 −4294 −4296 −5316 −5507 −2851 −989 −4418 −6132 −6118 110 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 88 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 111 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 89 1517 −3229 −5727 −6079 −5923 −3503 −5226 −5770 −5875 −6027 −5062 −4306 −4317 −5318 −5515 3379 −3101 −4447 −6127 −6104 112 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 90 −6077 −5086 −6532 −6846 2340 −6389 −2658 −5069 −6303 −4398 −4476 −5051 −6275 −5173 −822 −2095 −5956 −5209 −1911 4499 113 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 91 3609 −3185 −5808 −6157 −5905 −3465 −5219 −5724 −5874 −5992 −5019 −4286 −4281 −5312 −5496 −1323 −1317 −4399 −6126 −6106 114 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 92 −7065 −6301 −5432 3971 −7597 −5973 −6220 −8534 −6864 −7966 −7842 −6103 −6452 −6480 −6846 −7141 −7219 −8140 −6459 −7376 115 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 93 −1143 −142 −1816 −1265 −1849 −2885 2566 −3075 −1162 −3119 −2250 2224 −2976 549 1284 22 −1816 −2718 −3363 2668 116 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 94 −778 −3088 −6056 −5615 −3487 −5557 −4961 224 −5405 1064 −2368 −5256 −5471 −5166 −5368 −2258 −3463 3318 −4638 −4240 117 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 95 −7208 −6181 −7133 −7515 −7691 −6164 −6829 −8742 −7718 −8131 −8044 −7415 4335 −7586 −7182 −7628 −7478 −8268 −6469 −7677 118 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 96 −3985 −3461 −6668 −6345 −3798 −6525 −6482 −71 −6314 −256 −2502 −6194 −6189 −6129 −6427 −5920 −3970 3690 −5621 −5243 119 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 97 −3991 −3463 −6674 −6347 −3792 −6546 −6497 3037 −6321 949 −2491 −6205 −6196 −6129 −6433 −5941 −3974 2315 −5624 −5261 120 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 98 596 820 −1982 −395 −3866 −3091 4492 −3576 1175 −3535 −2649 −1753 −3199 −622 −1621 −809 −2098 −3166 −3702 −3080 121 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 99 −4049 −3520 −6722 −6389 −3668 −6582 −6459 3877 −6352 −540 −2381 −6253 −6204 −6079 −6422 −5980 −4029 80 −5503 −5203 122 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 100 494 −2807 −5440 −5132 −3532 −3878 −4109 −567 −4797 −3159 −2645 −4193 −4378 −4426 −4618 435 2115 2834 −4160 −3834 123 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 101 −1391 −3263 −5642 −5995 −5919 3758 −5222 −5713 −5850 −6009 −5071 −4317 −4344 −5310 −5512 −2913 −3136 −2419 −6119 −6090 124 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 102 2733 −2273 −4434 −3909 −2485 −356 −2923 92 −3584 −2375 114 −3392 −3815 −3262 −3500 96 1614 −788 −2968 −2627 125 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 103 −1857 −3322 −5407 −5758 −5957 −3576 −5181 −5791 −5725 −6045 −5116 −4303 4240 −5243 −5450 −2966 −1879 −4516 −6124 −6097 126 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 104 739 2166 −3053 −2548 −3830 −1155 −2652 −3482 −2430 −3664 −2855 −1084 1826 −2337 −974 1706 1974 −1611 −4015 −3529 127 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 105 −584 −1210 −1961 −1408 −3094 −2951 −1630 −2744 835 1117 552 −1676 −3038 1043 1139 627 1691 −2464 −3207 −2635 128 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 106 1269 −3316 601 −183 −3637 −503 2003 −3387 1102 −1799 −2405 470 −2910 662 532 −1029 223 −856 −3499 −237 129 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 107 2132 −334 −4404 −3773 −779 −3637 −2512 19 −1401 −320 −1123 −3274 −3687 −3007 −3189 887 −2030 2059 −2386 −2043 130 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 108 −1912 −3017 −1930 800 −3212 −2947 −1618 −2375 −1250 −2975 2135 −1653 −3035 3555 −353 −997 −562 30 −3281 −2693 131 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 109 329 −3316 547 853 −3637 −74 −461 −3388 648 −1400 −2405 927 −2910 2110 1126 −131 −960 −2938 −3499 −2817 132 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 110 1081 −3316 −485 −1 −655 −1502 −1475 −3387 492 −1817 −2405 2234 −2910 968 702 651 −218 −2938 −3499 −2816 133 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 111 −2379 −4629 −4310 −3233 −5439 3037 1554 −4835 875 −4562 −3855 −3199 −4471 −2131 1794 −3738 −3605 −1541 −4465 −4291 134 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 112 694 −3313 163 2013 −3632 −2820 −1479 −3381 1536 −804 −2403 −1457 −2914 −1020 1568 −887 −797 −2934 923 −2817 135 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 113 −323 1120 −4344 −3712 247 −3601 −2469 −240 −1332 2332 −1098 −646 −1255 −2955 −545 −2683 −2000 −679 −2352 1963 136 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 114 −4451 −3901 −7015 −6567 −3002 −6829 −6062 744 −6450 2011 1626 −6513 −6197 −5660 −6216 −6199 −4380 2735 −4809 −4848 137 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 115 −7502 −6257 −6956 −7316 −6597 −6224 5470 −8588 −7435 −7924 −7893 −7283 −6667 −7369 −6998 −7889 −7683 −8284 −6099 −6304 138 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 116 −7502 −6257 −6956 −7316 −6597 −6224 5470 −8588 −7435 −7924 −7893 −7283 −6667 −7369 −6998 −7889 −7683 −8284 −6099 −6304 139 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 117 −2651 −3259 −5623 −5970 −5916 −3527 −5205 −5778 −5810 −6031 −5076 −4306 −4338 −5289 −5482 2669 3129 −4469 −6111 −6072 140 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 118 −5212 −4579 −7571 −6972 −782 −7362 −5908 −2068 −6768 3255 319 −7107 −6304 −5429 −6189 −6759 −1324 −2952 −4373 −4576 141 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 119 751 −3208 −5779 −6119 −5853 3485 −5219 −5430 −5868 −5887 −4971 −4306 −4309 −5314 −5502 −2867 −3084 146 −6095 −6062 142 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 120 −3383 −5118 3723 1062 −5351 −3589 −2762 −5198 −2763 −5098 −72 −202 −4020 −2392 −1014 −3114 −1152 −4709 −5282 −4399 143 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 121 −4895 −5084 −6343 −6718 −7095 3848 −6267 −7596 −6999 −7455 −6893 −6032 −5861 −6658 −6640 −4594 −5331 −6556 −6371 −7085 144 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 122 −1856 −3327 2884 753 −3646 −2826 −1488 −1726 620 −3342 −2416 1379 −2921 −1029 −139 −1312 372 −850 −3510 −2828 145 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 123 −6226 −5136 −6586 −6947 4390 −6472 641 −5112 −6503 −4414 −4512 −5080 −6327 −5222 −5864 −5723 −6075 −5269 −1900 1459 146 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 124 −98 −3316 2335 695 −3637 292 939 −3388 −178 −1874 −294 347 −2910 212 621 527 −1782 −2938 −3499 −2817 147 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 125 −1969 −3424 499 −1237 −3741 −2909 4427 −3484 −460 −3434 −117 1728 −3018 −1130 −630 −531 −788 −3045 −3605 −2929 148 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 126 −4377 −3919 −6247 −5959 4054 −5797 −3218 415 −5594 −2233 453 −5033 −5613 −4783 −5225 −4975 −4289 −623 −2516 1996 149 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 127 130 −1996 −3693 −1282 1123 −1049 −2322 733 −2825 103 2119 −2892 −3550 −671 861 1302 −880 −1418 −2440 1409 150 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 128 −1093 −3427 −454 −139 −3756 −2910 1303 −3500 1564 −3437 −2518 1645 −3010 −1108 2875 −1838 −1051 −3054 −3596 −2924 151 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 129 −1644 2504 −5250 −4717 −2755 −4352 −3600 956 −4367 −2313 4736 −4165 −4476 −4020 −4204 −1910 −2754 −103 −3445 −3095 152 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 130 1364 −2018 −1276 −2999 1987 −3472 1052 −853 −2743 −1875 1530 −2833 −3531 −43 −2812 1185 −1970 −292 −2458 2041 153 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 131 999 −3546 −1981 1759 −3905 −3063 −1660 −3626 1724 −3544 −2640 −1700 −3148 927 1869 132 −2046 −3192 −3678 −3050 154 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 132 −511 −4187 −616 3053 −4478 −3254 1223 −4263 −1900 −4193 −3314 −1870 1602 948 −2474 −1151 −2588 −3799 −4366 −3610 155 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 133 −3940 −3424 −6611 −6275 1633 −6450 −6279 2805 −6229 −1337 −2513 −6102 −6133 −6047 −6330 −5822 −3923 2664 −5509 −5086 156 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 134 −1819 1338 −5951 −6288 −5880 −3460 −5233 −5688 −5905 −5962 −4992 −4303 −4277 −5338 −5504 825 3769 −4379 −6108 −6092 157 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 135 910 3533 −4896 −387 −2550 −4151 −3262 −192 −3997 −2235 −1707 −3863 −4252 −3669 −3869 −3287 819 2580 −3163 −2807 158 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 136 3291 −2359 −4831 −4349 −611 −2819 −3181 −428 −3975 −2530 −1885 −3628 −3933 −3599 −3794 −934 −1230 −69 −3155 −2824 159 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 137 175 −3308 −1756 −676 −3618 −2866 −605 −3352 −1093 −3318 −2408 −1512 −2960 3611 723 297 205 −1467 −3502 −2838 160 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 138 2656 −3129 −4534 −1425 −5015 293 −4142 −4719 −4239 −4955 −4101 −3700 −4103 −3992 −4393 288 2198 −886 −5249 −4951 161 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 139 −1370 −55 −449 −1452 −881 −3377 −2170 847 −1336 303 1215 1211 −3442 −689 800 544 −1766 390 386 1832 162 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 140 −4666 −4085 −7206 −6739 −2888 −7059 −6171 2059 −6623 2843 −1638 −6762 −6284 −5663 −6301 −6464 −4576 −17 −4746 −4868 163 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 141 −1965 −2646 −509 −1738 −2748 −3094 −1837 −1635 −1495 −2576 686 312 −3193 −1553 −2049 213 3394 −1410 −3014 −2300 164 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 142 1213 −3367 −1730 1318 −3673 −2897 −1585 −3409 −475 −3381 −2475 −452 2985 −393 −1684 −1836 −870 −656 −3571 −2900 165 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 143 1241 −3285 416 2538 −3591 −1180 −1489 −3330 −1076 −3295 892 −1469 −2921 1325 −1581 −1107 −1236 −1083 −3478 −529 166 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 144 −3916 −5618 −614 −2077 −6074 −3789 1523 −6152 −3669 −6012 −5421 4062 −4389 −3004 −4565 −3604 694 −5528 −6138 −5077 167 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 145 3567 978 −5981 −6297 −5716 −3512 −5246 −4854 −5908 −5616 −4816 −4347 −4320 −5358 −5517 −2879 −3081 −1006 −6042 −5983 168 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 146 2015 2840 −2231 −240 −2709 −2225 −1171 −2314 −690 −1157 −1780 −1893 −3145 323 −1136 −2012 1538 614 −2967 −2466 169 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 147 923 −3292 −301 1102 −202 −2823 414 −3345 −652 −3303 −665 −1462 −2916 436 145 858 1333 −383 −3482 1173 170 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 148 −4746 −6080 −2405 3890 −6602 −4417 −3936 −6827 −4165 −6572 −6094 −3175 −4990 −741 −4816 −4411 −4860 −6287 −6258 −5679 171 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 149 −4076 −3559 −6706 −6388 −414 −6496 −5943 3936 −6329 −2305 −2377 −6165 −6165 −5988 −6348 −5884 −4060 −353 −5056 −4492 172 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 150 −5479 −6001 3976 −3923 −7144 −5083 −4963 −7619 −5514 −7306 −6919 −4269 1563 −4832 −6140 −5307 −5677 −7002 −6429 −6542 173 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 151 −4157 −4830 −4021 −3534 −5941 −1516 −1890 −5459 −1622 −5102 −4460 −901 −4750 −2562 4104 −4087 −4040 −5106 −4880 −4739 174 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 152 −1230 −3448 −6531 −6232 −3676 −5893 −5997 −987 −6117 931 −2429 −5853 −5865 −5826 −6112 −5253 −3873 3499 −5328 −5018 175 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 153 −5088 −4460 −7490 −6961 −2671 −7311 −6082 2574 −6794 2731 −1438 −7088 −6341 −5551 −6284 −6778 −4945 −2519 −4528 −4735 176 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 154 −502 256 −1082 −2 −2047 −122 1156 −732 −1110 −1537 −2310 887 −2937 −589 2094 478 950 −79 −3420 −2767 177 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 155 1627 −292 −837 1625 −3365 −2085 −1537 −432 −1156 −2323 −84 −1541 −2960 886 −648 258 1201 −834 −3358 −2728 178 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 156 2052 3609 −4698 −4082 −2140 −3930 −2844 91 −3696 −879 2232 −3581 −3969 −3330 −3510 −1969 −2275 1429 −2699 −2353 179 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 157 −1253 −1905 −4366 −3734 −437 −3613 491 265 −3338 2361 −1105 −3241 −3663 −936 508 −2696 −2012 −1127 3469 −257 180 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 158 −150 −3304 −1696 409 5 −2820 282 −3366 1001 306 −2394 495 −2913 923 866 −6 1115 −2813 −3490 909 181 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 159 −539 −3316 −672 2553 −3637 −2818 1060 −3388 531 −3332 −1076 −1453 −2911 2520 −1033 −1299 −927 −2938 −3500 −533 182 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 160 −4547 −5064 −5790 −3852 −6075 −4958 2134 −5253 2618 −4829 −4196 −3620 −4840 137 3153 −4410 −4131 −5069 −4615 −4610 183 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 161 −4564 −5065 −5836 −3885 −6043 −4971 −2702 −5252 1617 −539 −4204 −3646 −4858 −2283 3786 −4432 −4151 −5075 −4624 −4616 184 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 162 −7208 −6181 −7133 −7515 −7691 −6164 −6829 −8742 −7718 −8131 −8044 −7415 4335 −7586 −7182 −7628 −7478 −8268 −6469 −7677 185 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 163 −3730 −4124 −6140 −6456 −6100 2458 −5821 −4478 −6441 −5830 −5367 −5254 −5201 −6048 −6124 −4044 −4165 3194 −6172 −6356 186 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 164 −1778 −4990 −6249 −6499 −1236 −6174 3804 −4988 −5932 −2253 −4410 −4962 −6148 −5061 −5496 −5454 −5708 −5098 −1956 4251 187 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 165 −4822 −4224 −7325 −6829 −2786 −7176 −6144 2946 −6695 2440 −1541 −6896 −6308 −5612 −6296 −6591 −4713 −1401 −4651 −4819 188 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 166 −728 −2491 −2396 −820 −529 −1480 2449 −2137 −1709 551 2170 1357 −3205 2107 −2090 −684 −1878 310 −2859 −2389 189 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 167 −4893 −4287 −7376 −6861 −2739 −7226 −6120 950 −6718 3118 −1495 −6949 −6313 −5580 −6283 −6641 −4773 −350 −4604 −4797 190 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 168 351 −3207 −5609 −5961 −5915 −3477 −5187 −5749 −5823 −6005 −5035 −4261 3981 −5265 −5483 391 −3074 −4422 −6127 −6096 191 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 169 858 18 −4361 −3729 −1853 −1566 −2476 1879 −3335 −1127 661 −3236 −559 −2961 −3152 1258 1322 965 −2355 −2012 192 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 170 −4148 −6239 4058 −1433 −6359 −3835 −3318 −6393 −3797 −6207 −5687 278 −4442 −3020 −4863 −3737 −4263 −5812 −6393 −5231 193 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 171 −3900 −3378 −6602 −6299 −4061 −6478 −6611 1567 −6289 −1856 −2736 −6137 −6191 −6262 −6484 −5878 −3892 3579 −5900 −5369 194 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 172 3057 −1041 −4882 −4509 −3304 −2259 −3576 −2874 −4191 −3213 −2525 −3711 −1138 −3845 −4086 −1345 823 −1048 −3757 1527 195 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 173 0 −3315 102 1168 −3636 −1401 2205 −918 931 −699 1006 757 −2910 468 1097 −203 −1782 −2937 −3499 −2816 196 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 174 1333 −2026 −3421 −2835 626 −3415 −2228 −786 1533 −311 1018 −2722 −3476 −583 −611 −85 1127 804 −2462 −2088 197 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9519 −10562 −894 −1115 −1074 −1929 * * 175 139 −3260 −427 1805 −3566 −2802 −1462 −869 479 −2409 −2353 −1443 2641 −419 −1554 −912 −494 −701 −3452 −2778 198 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9519 −10562 −894 −1115 −508 −1751 * * 176 992 350 −5269 −4703 −2721 −4602 3614 2819 −4367 179 −705 −4249 −4594 −4041 −4222 −3732 388 1547 −3433 −3068 199 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 177 −178 −3299 −681 2271 −3613 −1174 −1480 −516 −1064 −3312 −2391 809 −57 547 −1570 306 429 25 −3487 11 200 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 178 399 −2224 −2925 −2354 −2230 −3307 −2078 980 1150 −1510 −1411 −2407 2228 −2030 −290 −362 −381 1376 −2638 −2230 201 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 179 -295 −3330 69 −11 −3651 −2826 −8 −3402 −564 −3346 −2420 −957 3341 −246 5 −333 −378 −2952 −3514 −2830 202 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −6150 −894 −1115 −701 −1378 * * 180 688 −3299 527 732 −3619 −1525 −156 −2662 −53 −2113 −2388 −536 799 175 −1431 1104 1315 −176 −3483 −930 203 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9530 −10572 −894 −1115 −563 −1630 * * 181 1449 −3316 −279 901 −3637 −748 −545 −3387 −20 −1096 −98 500 334 858 −608 257 714 −2938 −3499 −839 204 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 182 687 −2468 −2431 −1872 891 −2446 −1867 −1089 671 −2363 −1637 −2047 2908 −665 −1235 104 −1342 −334 −2840 −375 205 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 183 −685 −2101 −1851 −556 556 −3395 −2193 587 −238 2459 −661 −2628 −1101 −544 −2634 −1241 −1951 −950 −2531 −2146 206 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 184 485 −56 −991 −917 −2512 −1566 −1871 174 −402 636 1452 −1100 1300 −94 −2120 −849 595 1051 −2835 −2371 207 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −193 −9550 −3014 −894 −1115 −701 −1378 * * 185 −904 −3125 814 351 −3425 −2682 1036 −1436 −43 1075 −1102 406 919 −357 −137 −1590 804 −382 −3320 −122 208 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9360 −10402 −894 −1115 −217 −2839 * * 186 56 −918 −892 1035 −3636 −1008 153 −3387 614 −877 −2405 −570 1345 177 1285 522 5 −1842 −3499 37 209 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 187 −17 −3306 −341 293 −3622 −2819 1297 −1375 −356 473 −2396 −556 862 1430 568 −616 739 −709 −3492 462 210 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −9550 −5406 −894 −1115 −701 −1378 * * 188 794 −3180 −543 432 −468 −2030 339 176 6 −3175 −48 −1475 2558 −1041 −670 10 −250 −1235 −3392 −2740 211 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9516 −10558 −894 −1115 −494 −1786 * * 189 64 368 873 593 −3597 −471 576 −2180 −1069 −261 184 −726 182 1713 −563 639 −501 −15 −3479 −2477 212 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −99 −9550 −3938 −894 −1115 −701 −1378 * * 190 −282 −512 −2828 −1434 −170 645 16 −254 −1356 87 −1328 −2315 −1611 408 −1561 2032 −65 241 −255 −436 213 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −486 −9453 −1814 −894 −1115 −1642 −557 * * 191 −3333 −5277 2899 −255 −5465 −807 −2594 −5412 −2900 −5268 −4626 2537 3758 −2270 −3810 −2978 1764 −4865 −5467 −4408 214 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −4 −8972 −10014 −894 −1115 −101 −3889 * * 192 −951 −3316 1033 477 −3637 −1345 −1476 −3387 1097 −3332 −2405 −1453 754 245 −875 1874 −500 490 −3499 −2817 215 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 193 −417 −3316 1110 1514 −3637 −2817 −107 −3388 432 −1539 −2405 179 1286 1023 −100 522 −377 −2938 −3499 −19 216 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −193 −9550 −3015 −894 −1115 −701 −1378 * * 194 1392 −3167 581 1107 −3486 −1501 −1331 −584 −912 −1717 −2256 500 −2765 319 1158 −199 1169 −2788 −3351 −2669 217 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9560 −10402 −894 −1115 −2161 −365 * * 195 1595 1189 −3929 −3322 −1791 −3401 −2300 −1325 −2989 1524 −859 −2959 −3477 1196 −2911 1054 −1271 533 −2283 −1933 218 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −94 −9360 −4016 −894 −1115 −2161 −365 * * 196 −121 −65 741 580 −3239 −2636 −1301 −2111 −376 1823 −2077 −207 −2728 854 487 −1548 −1579 82 −3191 −2543 219 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9269 −10312 −894 −1115 −167 −3191 * * 197 1478 −3294 −1703 641 −3604 −2823 −1483 −1763 1315 −1356 −2386 −1463 −2916 2142 −41 553 −122 −1071 −3483 −2807 220 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 198 2314 −1906 −718 −712 560 −1707 −2453 194 −3260 −80 2103 −3188 −3641 −2910 −3111 −2668 −1998 −582 −2361 1787 221 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 199 217 −2387 −5003 −4397 3415 −4270 −3192 1266 −4024 226 −314 −3918 −4273 −3642 −3838 −3377 −1261 1139 −2996 −2669 222 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 200 −407 −1064 792 263 −3592 −2825 −1482 584 598 −700 −174 455 −2917 618 1453 −305 733 −106 −3477 −2803 223 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 201 467 −3298 1205 1209 −3610 −1402 1716 −1284 686 −844 1509 −1460 −2914 170 590 −1728 −622 −1334 −3486 709 224 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 202 2225 404 −1284 −1624 −1979 −3481 819 1211 −2777 212 −1205 −2858 −3539 −1435 −1516 −1527 −478 1170 −2450 −2085 225 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 203 2320 342 −3573 −336 −775 −3468 −2290 −245 112 620 −1217 −2823 −1531 5 −2803 −2511 −225 659 −2462 −2094 226 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 204 116 −3316 709 1633 −3637 −2817 −1475 −1069 534 −3332 −2405 −93 −1403 458 1532 977 146 −2938 −3499 −2817 227 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 205 558 652 −822 955 −3633 −1383 734 −59 1665 −3329 −98 −1063 −2911 1062 984 407 −1079 −1383 −3497 −2815 228 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 206 −2061 46 −4377 −3743 592 −3605 −572 1161 72 2217 1211 −3242 −3656 −1516 609 −2689 −2002 −532 −2349 −615 229 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 207 61 −3237 −883 546 −1254 −2837 345 234 508 1837 −511 921 −2930 259 −580 −1071 −1789 −2840 −3442 −2781 230 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 208 924 −3316 −724 −184 −3637 −207 567 −3388 697 −1142 386 546 −1156 1439 −234 1558 −1009 −2938 −3499 −2816 231 — −147 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −1442 −5546 −712 −1428 −670 −701 −1378 * * 209 52 −2375 −69 90 −2692 −85 −546 −2440 662 −2389 −360 855 1027 −88 −635 1446 742 −1995 −2561 −1881 235 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394−88 45 96 359 117 −369 −294 −249 — −7 −8192 −9234 −894 −1115 −58 −4653 * * 210 2779 −3562 −409 −388 −3918 −45 −1741 −3674 −1369 −3624 −2712 −552 −3136 −1294 −763 1059 −852 −3218 −3798 −3103 236 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 211 −467 −494 −771 −1142 −3637 −1559 1915 −3388 2428 −3332 −2406 −519 −676 1055 1641 178 −1470 −2938 −3500 −2817 237 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 212 −282 −3316 24 −512 −1347 −1327 −1476 −3388 1479 −3332 −2405 −73 −2910 2026 2211 132 −35 −2938 −3500 −2817 238 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 213 −2295 −2108 −4597 −3977 −2068 −3876 −2766 398 −3594 −194 −233 −3503 3005 32 −3423 −2966 351 1867 −2632 −2291 239 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 214 1796 −1922 −4253 −3631 −1885 −1609 26 1264 −3262 −957 −1130 −3191 −3649 −2916 −3120 540 965 1745 −2385 −2039 240 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 215 −4311 −3769 −6913 −6502 −3174 −6745 −6161 −2536 −6411 1908 518 −6420 −6195 −5768 −6268 −6122 −4258 2011 −4984 −4945 241 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 216 −5108 −4472 −7528 −6958 −2625 −7385 −6070 1776 −6792 2969 237 −7116 −6334 −5507 −6260 −6802 −4951 −1315 −4493 −4743 242 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 217 3173 −3117 −5983 −5798 −4068 −4413 −5073 748 −5543 −3238 −2967 −4819 −4914 −5229 −5412 −3740 930 218 −5085 −4728 243 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 218 −5226 −5864 3782 −3755 −7088 1863 −4825 −7475 −5371 −7202 −6770 −4093 −5524 −4670 −6040 −5069 −5445 −6801 −6433 −6456 244 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 219 −738 17 −4321 −3691 2991 −3599 2990 −512 −3305 501 −1103 −330 −3650 532 −3135 −2680 −2002 −573 −2354 830 245 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 220 −3349 −4496 456 2379 −4509 −770 −2871 −4175 −2998 2186 1503 −2332 −4088 −2574 −3652 −3201 −3371 −3977 −4805 −4115 246 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 221 2046 −3272 −6399 −6083 −4010 −5884 −5965 1906 −5992 −2872 −2747 −5735 −5848 −5892 −6086 −5223 −886 −2656 −5546 −5049 247 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 222 −54 −3309 1635 35 −3627 −1897 2711 −960 −1059 −188 −2399 1803 −2912 1139 −862 −640 −998 −2929 −3494 −944 248 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 223 −3098 −3625 −5295 −4451 −5062 −3953 −3514 −1610 −2391 −4620 −4013 −3861 −4465 −3223 3855 1097 −3345 −1405 −4916 −4712 249 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 224 −480 −2547 −4958 −4418 3548 −4239 1638 −2053 −4012 224 874 −3760 −4269 −3548 −3778 −3337 −2703 −1443 1578 1997 250 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 225 −1425 −3364 −1163 −1183 −3687 2416 2428 −3435 361 −3378 −2455 198 −2954 2148 −503 −1375 −1834 −2987 −3543 −1963 251 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 226 1610 1808 −4498 −3864 −209 −3711 −2584 −474 −3463 1911 −572 −3356 −3753 248 −3264 −2797 −2095 1205 −2438 −2102 252 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 227 −137 −3313 −1692 594 −3633 −974 2438 −1592 545 −3329 −2403 −468 −2911 2586 778 −1725 765 −564 −3497 −2816 253 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 228 −1845 −3319 291 1799 −3640 −1094 691 −3390 1294 −3335 −2408 541 1027 1533 −1118 477 −726 −2941 −3502 −2819 254 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 229 944 −3302 −1186 1085 −1576 −2820 −1479 −3363 173 302 −2393 −72 −363 1636 105 −629 315 −179 −3489 42 255 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 230 −5010 −4399 −7433 −6871 442 −7248 −5952 −283 −6694 3020 −1400 −6963 −6282 −5474 −6195 −6634 −4864 860 −4463 −4655 256 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 231 −234 −3312 −1692 1749 −3631 −2818 1564 −2067 201 −2127 −2401 1286 −2911 2118 473 −1256 −29 −701 −3496 −254 257 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 232 888 −3317 716 566 −3638 −643 724 −3389 692 −2427 −2406 241 −2910 2471 1217 −1724 −874 −2939 −3500 −2817 258 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 233 −3693 −3374 −5859 −5351 2703 −5234 −3388 235 −4974 1594 −1643 −4724 −5090 −4323 −4673 −4368 −3609 20 4336 −221 259 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 234 399 −1954 −4475 −3843 −1913 −3692 −2570 1440 −3443 1468 1971 −3335 −3738 −3069 −441 −784 −2075 1886 −2433 −2091 260 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 235 1306 −3325 1068 1459 −3646 −2822 647 −3397 98 −3341 −2414 1705 −2916 827 −1572 −97 296 −2835 −3508 −2825 261 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 236 543 −3325 121 1683 −3646 −988 −1483 −3397 2259 −2243 −2415 113 −2917 1584 −390 −1732 −1791 −2947 −3508 −2825 262 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 237 −308 1517 85 −3053 −66 −830 −2314 −489 −2789 −1868 −1209 −2867 −3543 −2543 −840 −522 2825 1160 −2454 −2089 263 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −61 −9550 −4652 −894 −1115 −701 −1378 * * 238 337 −3271 −148 −809 −3592 984 912 −3343 611 −3287 −2360 1200 1515 1127 −507 67 −1088 −395 −3454 −2772 264 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9492 −10534 −894 −1115 −409 −2017 * * 239 −3691 −3358 −6072 −5469 1503 −5448 −4297 2852 −5134 1057 2147 −5106 −5208 −4503 −4862 −4598 −3608 880 −3708 −481 265 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 240 569 −3220 −5550 −5905 −5918 −3487 −5180 −5756 −5803 −6011 −5045 −4258 4076 −5253 −5476 −1468 −3086 −4433 −6126 −6090 266 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 241 -1384 1643 −4437 −3803 102 −3644 3614 1059 −3400 −723 −1119 −3288 −3692 −3022 −3201 −2729 −2034 1693 −2368 2105 267 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 242 3136 −3152 −5586 −5725 −5563 −3456 −4957 −5305 −5471 −5590 −4683 −886 −4246 −5008 −5243 865 974 −594 −5821 −5708 268 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 243 −836 −93 −2326 −1794 −3700 −1392 −2039 −3389 −1698 −3474 −2615 405 −1211 1816 −2162 352 3080 −1366 −3746 −3163 269 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 244 −1184 −4148 −6981 −6387 −1813 −6444 −5279 −2010 −6105 3019 2579 −6214 −5880 −5098 −5677 −5700 −1428 −2745 −4172 −4275 270 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 245 −1020 −2014 −3864 −3270 −2004 −3515 −2414 −1547 −2980 1507 2096 636 366 −2705 −2977 2053 −1046 −363 −2485 −2128 271 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 246 −2069 −1896 −4408 −3773 281 −3616 −2485 −493 −3369 995 3825 −3260 −663 −2992 −3171 −1035 372 −1308 3429 −240 272 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 247 826 −3476 −5131 −4529 −6095 3602 −5206 −5975 −5789 −6195 −5270 −4344 −4497 −5255 −5574 −3119 −3346 −4687 −6176 −6199 273 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 248 −6970 −6153 −6729 −6584 −7461 −6082 −5807 −8068 4059 −7520 −7222 −6515 −6455 −5828 −4943 −7177 −6958 −7760 −6247 −7071 274 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 249 −873 1079 −5776 −6006 −5825 2990 −5126 −5636 −5701 −5893 −4930 −212 −4263 −5186 −5397 1735 523 −4358 −6047 −5999 275 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 250 1542 −2277 −4881 −4283 −2339 −4128 −3089 1815 −3911 1521 −1514 −3790 −4166 −3556 −3737 −733 −780 1446 −2939 −2588 276 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 251 −4845 −4284 −7252 −6672 3152 −6960 −5694 539 −6456 1931 906 −6664 −6139 −5338 −5997 −6278 −4706 1097 −4367 −4531 277 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 252 −1971 −5354 3059 −727 −5565 −3639 −2884 −5445 −1144 −5330 −4583 2612 1122 −2527 −3750 −376 −3578 −4935 −5517 −4577 278 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 253 −7065 6301 −5432 3971 −7597 −5973 −6220 −8534 −6864 −7966 −7842 −6103 −6452 −6480 −6846 −7141 −7219 −8140 −6459 −7376 279 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 254 −411 −3316 −1690 1383 −3637 −1391 1016 −3388 418 −3332 62 −44 −2910 1471 606 984 1557 −2938 −3499 −2817 280 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 255 −1041 −227 −320 −1158 −3595 −2825 3447 −3337 −507 −107 −2381 1617 −2918 1232 −1577 1053 69 −2903 −3479 −2805 281 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 256 192 −3308 −549 642 −2093 −2838 −1505 −3358 −1094 −3319 −2403 −1210 3279 −1050 −28 324 −1343 −2924 −3501 −1377 282 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 257 −951 −153 −2018 −1465 −1003 1296 2330 −2601 −1362 −878 281 1830 −3057 1451 204 −337 −1832 −2348 −3128 1181 283 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 258 −6230 −5137 −6592 −6954 3971 −6476 −2652 −5112 −6509 −4413 −4512 −5081 −6329 −5223 −5868 −5725 −6078 −5269 2517 3059 284 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 259 911 −2023 −4568 −3940 −1992 −3788 −2674 2132 −3544 774 1818 −3434 −3829 −3170 −3350 −2878 339 1541 −2530 239 285 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 260 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 286 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 261 −2623 −2441 −5057 −4504 −2610 −999 −3375 2409 −4149 −1250 −1757 −3980 −4328 −3808 −3992 −1754 2853 988 −3242 −2888 287 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 262 −6235 −5152 −6598 −6959 1041 −6461 −2688 −5132 −6524 −4432 −4532 −5109 −6332 −5251 −5886 −5746 −6091 −5291 −1935 4799 288 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 263 1571 −2667 1056 −1648 −2772 −3050 −1767 −277 −1537 −2592 −1831 1396 −3138 −1461 −1960 1566 183 155 −3013 −413 289 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 264 278 −3309 −5537 −5899 −5999 3635 −5237 −5851 −5877 −6093 −5136 −4331 −978 −5317 −5556 −2957 −3182 −4530 −6156 −6168 290 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 265 2101 −3312 909 502 −3629 571 −1483 −667 738 −3327 −2403 −358 −2916 −1024 −1572 369 −1453 −1436 −3498 −2818 291 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 266 2474 −1920 −1503 −3536 −46 −469 −2432 870 −3183 380 −1121 −1486 −1544 −2851 −3069 −1452 −794 619 −2374 −2026 292 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −32 −5546 −10592 −301 −2408 −701 −1378 * * 267 940 −3384 −4043 −1555 −5926 −3522 −4705 −5769 −5171 −5978 −5062 −3830 −4307 −4624 −5229 3465 −3180 −4526 −6109 −5888 294 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 268 1107 −3316 1180 1599 −3181 −1993 760 −3388 −274 −3332 −2405 −1452 −149 606 219 841 187 −2302 −3499 −2817 295 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 269 217 662 1204 1555 −3617 −65 −1481 −1370 −22 −2071 −2394 −1459 2376 −515 −1571 −702 −1785 −2921 −3490 −2811 296 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 270 603 −3315 664 269 −1112 −1083 126 −3386 −1 −1799 −2404 927 398 855 −750 860 −167 −1439 −3499 1485 297 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 271 −1381 −2813 −5640 −5127 −3119 −5045 −4203 1362 −4841 664 −2125 −4710 −5010 −4557 −4742 −2541 540 −3072 −3985 −3601 298 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 272 −2581 686 −2767 −2090 −1463 −3554 −2021 −3357 2221 −1714 −2639 −2277 −3603 585 3132 −2559 −1027 −3099 −3650 −573 299 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 273 374 −3318 1400 1763 −3639 −2818 −107 −3390 1273 −3334 −2407 890 −2911 1294 −371 73 −928 −2940 −3501 −2818 300 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 274 1677 −499 −4359 −3726 −146 −3603 957 88 −3331 194 −287 −3233 −3653 −614 1314 −2685 −844 584 −2350 2261 301 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 275 −3861 401 −6555 −6239 −4050 −6401 −6447 3335 −6213 −2806 589 −6058 −6136 −6178 −6390 −5781 −3852 2341 −5814 −5285 302 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 276 −997 −5914 1946 3305 −6053 −3757 −3147 −6008 −355 −5854 −5225 200 −4315 −2825 −4383 −3549 −3997 −5464 −6052 −4977 303 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 277 −975 87 879 1415 −3657 254 152 −3408 −588 −3352 −2426 2464 −2925 1765 −1584 −82 −1801 −2958 −3520 −2835 304 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 278 2791 337 −5913 −6176 −5634 −3462 −5126 −5343 −5793 −5672 −4761 −4279 −4266 −5246 −5422 −2292 −665 −594 −5905 −5836 305 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 279 −1658 −5883 3903 −130 −6247 −3788 −3286 −6237 −3720 −6082 −5511 −2412 342 −2982 −4729 −3635 −1287 −5629 −6288 −5166 306 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 280 1101 3805 −4419 −3785 975 −3629 −2502 −483 −3382 −734 −687 −3273 −3678 −3007 −741 −2714 578 1848 −2369 −2026 307 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 281 −1656 887 −4944 −4348 −2394 −4209 −3163 2406 −3980 1724 −1559 −3862 −4234 −3629 −3809 −1514 −79 1636 −3007 −2653 308 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 282 −4289 −3745 −6903 −6503 −3226 −6748 −6224 3165 −6423 1629 −387 −6422 −6208 −5816 −6305 −6131 −4240 1281 −5048 −4991 309 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 283 −1916 2920 −2174 −1619 −2780 −89 159 −2393 −271 −500 1182 1633 −3123 −1432 −1931 1312 1552 −2179 −3014 −2501 310 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 284 564 −2845 −5687 −5174 913 −5123 −4264 2749 −4894 −668 −2122 −4772 −5064 −4607 −4796 −4292 −820 2228 −4028 −3647 311 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 285 −4367 −4772 −4993 −5379 −6814 −3794 −5649 −7165 −6214 −7111 −6453 −558 −5482 −5753 −6195 −4572 −4792 −6082 −6315 −6679 312 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 286 1372 −2567 −5041 −4660 −3101 −1348 −3550 −2424 −4300 −1150 −2316 −3799 −731 −3918 −4116 −2826 843 3068 −3598 −3278 313 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 287 −2187 −3542 −832 −1507 −3888 −3136 −1708 −3586 2345 −940 −2641 −805 −3213 1327 2536 −2077 −2107 −417 1638 −3074 314 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 288 −5078 −4474 −7160 −6720 3570 −6878 −4562 −766 −6448 1849 301 −6279 −6146 −5256 −5923 −6168 −4919 −3065 −3545 1577 315 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 289 −1744 −3249 −6310 −6036 −4033 −5420 −5761 986 −5903 −2939 −2797 −5498 −5599 −5745 −5934 −4759 3494 1497 −5466 −5007 316 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 290 −4158 −6242 4095 −546 −6365 −3843 −3327 −6042 −3806 −6215 −5696 −1885 −4450 −3029 −4871 −3747 −4273 −5821 −6394 −5239 317 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 291 −2070 −1922 −4254 −3630 −468 −3600 −2466 −695 −3261 −1079 1424 −118 −3652 −2915 −3119 1722 2829 54 −2381 −2035 318 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 292 821 −1930 −4227 −3608 −1895 −891 −2467 2192 −3245 283 −1138 506 −3648 −2904 −3113 206 1066 834 −2393 −2047 319 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 293 −2668 −3276 −5605 −5952 −5919 −3542 −5207 −5786 −5804 −6039 −5089 −4315 −4352 −5291 −5481 960 3838 −4484 −6108 −6067 320 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 294 2880 −3177 −5884 −6221 −5901 2240 −5227 −5724 −5894 −5986 −5010 −4292 −4276 −5322 −5506 −117 −803 −4042 −6123 −6108 321 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 295 −792 −1903 −4289 −3661 642 2923 −2459 −951 −1279 −383 65 −3202 −3645 −2926 −3122 −37 −1999 −60 −2358 200 322 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 296 −6244 −5180 −6612 −6970 4536 −6435 −2762 −5155 −6554 −4451 −4554 −5167 −6339 −5306 −5923 −5789 −6115 −5326 −2010 −108 323 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 297 −2709 −3368 −4408 −4413 −5703 −3547 −4241 −5468 −3824 −5593 −4708 −951 −4265 −4032 136 2835 2650 −4395 −5693 −5486 324 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 298 −1590 −4003 −5042 −4704 −164 −5207 3683 −3707 −4354 −3626 −3266 −4166 −5214 3419 −4346 −4319 −4107 −1372 −2031 2271 325 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 299 −1007 −3318 43 −88 −3639 361 2154 −545 1262 −3334 −2407 −333 −2911 2444 1416 −1256 −1784 −2940 −3501 −2818 326 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 300 −2796 −2580 −5101 −4514 2679 −4367 −3125 2625 −4130 1453 −1399 −993 −4363 −3685 −3915 −1538 −2738 −1692 −2818 −2341 327 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 301 112 −309 2263 −654 −3626 −2819 −1478 −2196 506 −2422 −1272 859 1275 −1020 −1567 50 1375 −2929 −3494 −2814 328 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 302 189 −158 214 107 −3629 −2818 −1477 787 969 −2703 −453 −702 1571 1092 687 −977 902 −2931 −3495 −2814 329 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 303 1004 −3317 1862 1925 −3637 −1725 −1477 −3387 −482 −1441 −2406 907 −1114 −1018 −694 269 −1784 −1323 −3500 −2818 330 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 304 −848 −3317 726 −601 −3638 −1747 −211 −3388 1710 −333 −2406 1661 −1114 1774 1675 −377 −1782 −1440 −3500 −2817 331 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 305 −2061 −2331 −4385 −3751 −81 −2263 −2476 −727 −3350 −388 1592 93 −3656 359 −3158 −2690 2045 1937 −2348 291 332 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 306 −3867 −3392 −6454 −6049 −1237 −6150 −5473 3598 −5904 −758 −2249 −5789 −5877 −5564 −5867 −5440 −3835 1567 −4818 −1105 333 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 307 −2074 −3552 2839 1924 −3858 −2961 520 −3612 −1309 −3562 1958 −232 −3098 −1237 −1831 −366 −807 −3168 −3739 −979 334 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 308 −1518 −3380 −6459 −6049 −424 −6156 −5626 3261 −5910 1046 −2242 −5813 −5881 −5585 −5884 −5449 −3824 1463 −4979 −4685 335 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 309 −508 −3405 −908 −1211 −3730 995 2701 −3477 −56 −3417 −2496 1605 −2987 2778 210 −1812 −1873 −3030 −3580 −2902 336 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 310 −1852 −3217 −461 −1194 −3494 −1352 2085 −926 −1105 −964 −2317 −413 2944 999 481 −932 −866 −1795 −3427 −658 337 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 311 −1845 −3306 383 546 1790 −2820 2882 −3370 −596 −3320 −2397 926 −2913 569 −303 −1727 −65 −2926 226 2201 338 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 312 670 −3302 427 753 −518 −1028 1571 −1286 −1062 −1270 −2393 565 −2913 1385 −195 291 721 −1425 −3489 466 339 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 313 1627 459 −4403 −3768 1296 −1137 −2485 245 −3365 183 −1097 −3256 −3663 −2990 −3168 1074 −1000 1613 −2353 −2011 340 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 314 −1864 543 −1795 −1245 −3385 −2870 −1537 −1803 2267 −3122 −36 580 −2961 −384 2137 906 −690 −1120 −3370 −577 341 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 315 −269 −2686 −5434 −4878 −1053 −4793 −3824 2508 −4555 314 1042 −4441 −4760 −4218 −4413 −3933 −1375 2521 −3603 −3252 342 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 316 1002 −3281 239 −655 102 2322 −1487 −580 −46 −3290 −1597 1008 −1011 −151 −1578 −1734 −862 −2894 −3474 −2801 343 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 317 −1044 −3326 2472 679 −3647 1226 729 −3398 −699 −3342 −2415 867 −2917 266 −6 −157 −447 −2948 −3509 −2826 344 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5365 −10592 −275 −2525 −701 −1378 * * 318 −229 −3316 −1693 436 −3637 −2819 291 −3387 1725 −3332 −2406 −454 −2912 2108 193 −635 1704 −18 −3500 −2818 346 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 319 −666 −492 −1067 −2114 −1045 −3226 −1977 909 704 −1292 −1502 60 −2106 405 −722 −961 1049 1623 2972 −806 347 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 320 −630 −1913 −1273 −3600 3438 −3585 −2446 50 −3234 −1762 54 −683 −3637 −2891 −3098 −1587 −1361 −848 −2366 2790 348 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 321 1199 −3312 −362 544 −3631 −178 −1478 −3380 −1060 −3327 −2402 738 1276 −236 −1567 1104 1043 −1066 −3497 −727 349 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −45 −9550 −5105 −894 −1115 −701 −1378 * * 322 −308 −4084 587 108 −4380 1660 −2069 −4163 −1802 −4093 −3209 461 2505 1772 −2371 −1264 −2491 −3698 −4266 −3516 350 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9508 −10550 −894 −1115 −462 −1868 * * 323 −1953 −3366 −6460 −6062 29 −6175 −5701 2797 −5936 1043 −2312 −5830 −5906 −5649 −5934 −5476 −3817 2327 −5069 −4746 351 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 324 701 −3248 −99 834 −3539 −2836 −1498 −3268 −1091 −1842 −2346 248 2031 −1049 −1593 970 951 −123 −3451 −524 352 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 325 −1410 −3260 −5921 −5299 −184 −5253 −4088 778 −4945 1397 4400 −4914 −5049 −4326 −4669 −4388 −482 −2158 −3550 −3409 353 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 326 1285 −3315 −260 721 −3635 258 −1476 −962 1063 −1269 −2404 −194 688 423 612 −204 −1782 −747 −3498 −2816 354 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 327 494 −3316 2832 717 −3637 −1309 −527 −3387 −766 −3332 −293 −863 −2910 1379 −298 −72 −1279 −1790 −3499 −2817 355 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 328 2576 −2180 −4731 −4107 −1084 −3965 −2855 −848 −3717 1283 −275 −3612 −3990 −3329 −3521 −1720 −2319 1410 −2685 −2357 356 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 329 −4546 −3981 −7101 −6645 −2951 −6937 −6119 1480 −6530 −2638 −366 −6626 −6239 −5665 −6259 −6322 −4467 1758 −4785 −4864 357 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 330 768 −3316 878 1060 −3637 −2817 826 −1505 1346 −3332 −2405 174 −2910 1702 −1081 367 24 −1011 −3499 −2816 358 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 331 2281 −3237 −1732 415 −3523 419 220 −376 −431 −3238 −2335 −1487 −2930 609 −790 −667 634 −418 −3441 −1922 359 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 332 −5028 −4411 −7472 −6930 −2665 −7297 −6081 −1235 −6765 3214 −1426 −7047 −6323 −5536 −6268 −6718 −4890 317 −4529 −4745 360 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −265 −9550 −2586 −894 −1115 −701 −1378 * * 333 568 −3075 −494 429 −3377 −2627 2895 −2705 −875 −28 −117 538 −2719 −244 259 944 268 −853 −3259 −1366 361 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −2418 −9288 −302 −894 −1115 −2450 −292 * * 334 −777 −2314 2263 367 −2610 −1352 −308 −2393 −92 −2339 −1481 900 −1641 1579 −673 1108 −758 −1939 −2526 −1767 362 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −−2052 −406 −7930 −72 −4366 −4400 −70 * * 335 −1817 −1401 −4122 −3669 −818 −3836 −2960 3166 −3384 1220 314 −3462 −3558 −2957 −3280 −3105 −1773 784 −2289 −2053 364 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −2052 −406 −7930 −72 −4366 −4400 −70 * * 336 −1425 −2343 −1566 −736 −2851 −2127 3928 −2415 1528 −2219 −1486 −804 −2140 118 −1794 −1322 −1224 −2137 −2168 −1869 366 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −18 −6888 −7930 −894 −1115 −779 −1262 * * 337 −585 −2852 −357 1803 −3173 −2352 −1011 −2924 193 −2868 −1942 −459 560 1915 1554 −358 423 −2474 −3036 −2353 367 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −4 −8921 −9963 −894 −1115 −499 −1775 * * 338 −658 −2338 374 −477 −2387 −3029 −1759 995 356 1590 197 1348 −3107 −1542 −2011 −1493 −696 −56 −2712 −2250 368 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9403 −10445 −894 −1115 −1951 −432 * * 339 651 2565 −304 391 311 −807 −1432 −2906 113 47 −71 −914 −945 −1008 126 −92 1163 −1212 −3224 −1155 369 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9403 −10445 −894 −1115 −788 −1249 * * 340 855 −3195 −410 1066 −3484 −1895 −1447 796 432 381 1071 −1026 −1154 405 −36 −1162 335 −400 −3398 −2734 370 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −35 −9486 −5454 −894 −1115 −392 −2070 * * 341 −36 −3292 1417 918 −3613 −1554 526 −2214 677 −3308 −2381 −514 99 684 482 952 910 −2914 −3475 −2793 371 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9519 −10561 −894 −1115 −1081 −923 * * 342 −244 1040 −1072 −1204 466 −486 823 −1937 −1882 1653 −319 −2160 −456 −1120 624 −1285 −1876 −1790 −2723 2228 372 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −33 −9519 −5562 −894 −1115 −1081 −923 * * 343 701 −703 642 −128 −3586 722 952 −742 1116 −3282 −853 556 −2863 323 124 82 −534 273 −3450 −2768 373 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −107 −9489 −3839 −894 −1115 −401 −2044 * * 344 262 −3195 −694 260 1240 −594 127 1133 −707 −2472 −2289 1639 −693 691 −664 −276 −3 −445 −3390 −1187 374 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9451 −10493 −894 −1115 −318 −2337 * * 345 125 −3316 −619 969 −3637 −422 1114 −350 1730 −2129 −2405 522 −2910 630 835 −137 −131 −812 −3499 −2816 375 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −72 −9550 −4403 −894 −1115 −701 −1378 * * 346 536 −2610 −2122 −68 108 −1068 105 −2319 −1454 −1611 −1764 −438 1293 −1379 −1879 1204 −172 −552 3942 −2434 376 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9481 −10523 −894 −1115 −379 −2112 * * 347 405 −3229 744 −505 418 −2839 1259 −1718 −1098 −465 −2329 −1490 1869 1360 −353 333 −267 −2831 −3436 349 377 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 348 151 −3226 −647 −228 538 −935 636 241 745 −368 −197 −426 1304 477 −386 −1750 791 −1863 −3434 506 378 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 349 531 −979 −681 137 −993 186 −1528 −3105 −1141 −1335 −2259 −1528 2491 −223 −751 926 −279 −396 −3378 −2740 379 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −25 −9550 −5957 −894 −1115 −701 −1378 * * 350 699 −496 −857 947 −995 −2195 −1516 −269 197 −1236 −108 −41 −648 1131 −1046 −358 886 766 −3348 −71 380 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −220 −9527 −2834 −894 −1115 −988 −1012 * * 351 632 −1789 −1615 −2960 144 −3309 620 1093 −153 527 172 −2732 398 −2414 −563 239 −1698 433 −2234 1925 381 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −30 −9310 −5717 −894 −1115 −305 −2393 * * 352 412 −764 −266 682 −577 −2832 493 −76 536 −355 −1251 −685 989 256 −1609 205 679 132 −3326 −1304 382 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9504 −10547 −894 −1115 −1227 −804 * * 353 731 −3281 −359 795 −3602 −1089 636 −3353 84 −3297 −2370 −699 2030 506 1324 237 −882 −1451 −3464 −1401 383 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −588 −9504 −1586 −894 −1115 −1227 −804 * * 354 131 −2851 −188 366 −3172 −475 0 −2922 682 −1961 −1940 −988 2819 316 −93 −128 −1318 −2473 −3035 −2352 384 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −124 −8921 −3641 −894 −1115 −2727 −236 * * 355 801 −2783 −81 1240 −472 −1325 −966 −2841 728 −404 −537 −945 −191 994 5 −36 201 −81 −2972 −2294 385 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −576 −8852 −1613 −894 −1115 −1706 −528 * * 356 757 −2227 −316 −674 −232 −1343 1335 575 −284 −422 −46 −25 166 894 −1045 668 506 −1767 −2506 −399 386 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −6 −8582 −9624 −894 −1115 −3259 −159 * * 357 101 −2637 −198 870 14 −1188 1496 −2700 1104 −1412 138 582 −667 −354 55 −434 1227 −651 −2824 −2144 387 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −5 −8624 −9667 −894 −1115 −74 −4331 * * 358 133 −1714 754 1380 −3636 −664 −581 −2447 336 −1337 −2404 612 1593 513 −48 −818 −358 −622 −109 −2816 388 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 359 436 −2977 −705 602 725 −2916 −1594 10 535 −958 −2103 −1623 1436 689 −897 −1157 −948 437 −3246 1547 389 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 360 205 −3251 −1068 1390 820 −1557 1174 −1360 −1087 −97 −2348 −717 678 753 263 −195 317 −1059 −3452 −150 390 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 361 267 −2930 −1931 407 −340 −1043 980 −1173 −640 61 −2060 442 2158 −100 −1751 −550 −299 311 −3209 −668 391 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −244 −9550 −2696 −894 −1115 −701 −1378 * * 362 93 −3130 232 357 −3451 −779 −415 −3201 −213 −3146 −2220 −1268 2376 1918 −797 537 −708 −1639 538 −2631 392 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −743 −9309 −1319 −894 −1115 −2374 −309 * * 363 −6 −2504 172 635 −2777 −1401 770 −220 −407 −2499 −387 −797 −50 1315 −905 604 1000 837 −2716 −630 393 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −6 −8572 −9614 −894 −1115 −3240 −161 * * 364 328 −2646 73 1229 −2966 77 795 −1751 −51 −2661 −1735 38 1698 −3 −895 1071 −622 −2267 −2829 −2147 394 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −2431 −1634 −1022 −121 −3635 −385 −2095 * * 365 598 −2843 753 134 −3164 53 407 −2914 1096 −2859 1932 400 −627 −91 621 1087 733 −2465 −3026 −2343 397 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −4 −8910 −9952 −894 −1115 −202 −2937 * * 366 369 −3283 2236 955 −304 −742 948 −3355 586 −3299 −2372 632 −749 396 −1063 −36 121 −2905 −3467 −1403 398 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −33 −9507 −5550 −894 −1115 −459 −1876 * * 367 1320 −3296 1066 1001 −3617 977 −112 −3367 −526 −3312 −2385 −201 −2889 1749 −1543 −423 330 −1596 −3479 −2796 399 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −34 −9520 −5517 −894 −1115 −513 −1741 * * 368 535 −2287 −2680 −191 −1076 −919 −1963 −824 486 −280 1374 −2225 2628 −1837 −1154 −860 156 −704 −424 −2255 400 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9519 −10562 −894 −1115 −508 −1751 * * 369 −4990 −4377 −7428 −6876 −749 −7259 −6008 1154 −6710 3030 −1420 −6977 −6295 −5503 −6224 −6655 −4849 −26 −4502 −4705 401 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 370 −1879 −3300 1041 −1190 −3604 −2852 −1519 −3340 −340 −2111 −2399 697 −2949 −1067 −663 981 2931 −1464 −3498 −2829 402 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 371 −4631 −5070 −3965 −3822 −3291 −4894 1160 −5212 −2442 −1423 −4417 −3081 −5130 4412 −2463 −4521 −4541 −5157 −3728 −2784 403 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 372 403 656 1535 1731 −3638 −2817 1362 −3389 1026 −3333 −2406 −262 −2911 1509 −58 −274 −1783 −2939 −3500 −2817 404 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 373 290 −3297 151 −364 −3609 −1155 566 −647 −1065 −3309 558 1242 −2915 −147 1886 −604 1462 −1381 −3485 611 405 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 374 −4550 −3991 −6555 −6115 3483 −6094 −3952 −526 −5787 1486 −1666 −5510 −5720 −4903 −5399 −5291 −4341 147 −3162 2158 406 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 375 −6168 −2253 −6583 −6932 2367 −6440 210 −5091 −6474 −4403 −4494 −5081 −6310 −5219 −5847 −5699 −6032 −5244 5850 −719 407 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 376 −781 −3317 345 −260 −3638 −1401 2233 −3388 996 −3333 −2406 582 543 2722 −45 −624 −1782 −2939 −3500 73 408 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 377 601 −2596 −2257 217 −2677 −3072 1005 −512 −1582 −992 2164 −1913 −3153 1887 1630 −2023 966 −1012 −2945 164 409 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 378 −4398 −3881 −6913 −6419 −642 −6623 −5696 2553 −6243 807 3319 −6304 −6049 −5441 −5971 −5933 −4318 1767 −4584 −4609 410 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 379 839 1027 −1737 649 −3509 −1610 −1503 −3233 −1099 −1517 −57 −98 −2932 3122 −277 −55 −1790 −2828 −3435 751 411 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 380 178 −3318 1699 −489 −3639 490 2731 −3390 −70 −3334 −2407 1139 −2911 147 1018 −691 −3 −2940 −3501 −2819 412 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 381 665 95 −4424 −3799 3791 −1077 1059 −1457 −3403 −783 −1166 −3290 −3716 −3027 −3215 −2749 −2071 −666 −2332 65 413 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 382 −4973 −4386 −7371 −6793 −875 −7113 −5796 1621 −6587 2985 −1369 −6833 −6212 −5392 −6090 −6463 −899 −2622 −4385 −4555 414 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 383 −718 −3321 −267 211 −3643 −1896 43 −3393 1228 −2312 −2410 −1457 −2915 2595 2216 −1729 490 −2943 −3503 −2821 415 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 384 283 −3376 −679 1631 −3693 −1166 −521 −3443 −598 −1766 −2468 −1491 3057 −547 −1634 −476 −1845 −2996 −3561 −2876 416 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 385 −3601 −5430 2293 −1155 −5635 2377 −2920 −5519 −803 −5396 −4664 2475 −4145 −2567 −1437 −1970 −3640 −5009 −5580 −4633 417 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 386 −4138 −6262 −3869 −485 −6355 −3822 −3301 −6381 −3781 −6195 −5674 1640 −4429 −3001 −4853 −3723 −4251 −5803 −6396 −5219 418 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 387 −3868 −3353 −6562 −6248 −4046 −6415 −6467 2771 −6223 −692 −2731 −6070 −6144 −6186 −6401 −5797 −835 −2931 −5820 −5295 419 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 388 −4103 −3569 −6763 −6412 −3548 −6628 −6401 2500 −6368 1930 −2265 −6293 −6206 −6008 −6391 −6021 −4075 2175 −5381 −5158 420 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 389 −1854 −3484 −6497 −6036 333 −6136 −5386 2014 −5852 2206 −1944 −5798 −5811 −5360 −5722 −5401 −3897 1893 −4650 −4470 421 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 390 3030 −3151 −5867 −6132 −5642 503 −5119 −5364 −5779 −5682 −4769 −4271 −4266 −5234 −5417 −1854 1009 1015 −5910 −5840 422 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 391 −4321 −6263 2878 3169 −6472 −3985 −3485 −6543 −3968 −6343 −5837 −2599 −4587 −3195 −5010 −3915 −4435 −5970 −6403 −5381 423 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 392 −126 −2450 −2873 −2327 −2552 −3319 −2181 −1382 −2146 −1342 −1698 −276 −3444 3984 −2445 −2341 −172 −413 −2931 −2505 424 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 393 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 425 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 394 −2633 −3242 −5534 −5866 −5777 −3512 −5127 −4621 −5651 −5926 −5012 −4266 −4320 −5208 −5358 −1912 −4033 −4404 −6021 −5883 426 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 395 1763 1191 −5942 −6273 −5887 −3460 −5231 −5706 −5900 −5971 −4997 −4300 431 −5332 −5504 3077 −3046 −4387 −6111 −6096 427 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 396 1173 1989 −3754 −3157 3016 −979 492 −1519 −2879 −1085 −1204 430 −3572 −2616 −2901 318 −1993 −1428 −2436 980 428 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 397 −53 −2388 −4783 −4246 3995 −3925 −2682 −506 −3857 −2282 −1673 −3619 −4065 −3443 −3657 −615 −1216 −1872 −2399 1378 429 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 398 145 −3184 −5785 −6136 −5912 3603 −5218 −5738 −5885 −5998 −5022 −4280 −4279 −5309 −5507 −1463 −576 −4403 −6132 −6115 430 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 399 2534 −1986 −4112 −3812 −1952 −3658 −2585 529 −3425 897 1819 −3317 −3731 −3059 −3250 610 −1392 −1362 −2471 −2130 431 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 400 2407 456 −4316 −3692 −1885 −220 −2486 −406 −3311 −1779 345 −3221 −3657 −2954 −3149 1254 −1819 35 −2386 1790 432 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 401 1448 −3306 1885 −29 −3622 141 −1478 −1331 −454 −834 −2396 −411 −555 −369 −1567 183 −858 −1997 −3492 −2812 433 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 402 −1088 −2049 −4563 −3929 −1930 −3788 2034 1160 −3530 2211 2299 −3429 −3822 −469 −3332 −2875 −2169 481 −2500 −2172 434 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 403 356 −3285 −1709 −1160 −577 −2098 132 −1707 535 −3295 −2378 −962 1288 −340 2765 313 −478 −2899 −3477 −2803 435 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 404 −5304 −4636 −7665 −7057 698 −7536 −6043 −815 −6875 3109 1688 −7277 −6363 −5472 −6265 −6968 −5114 −2978 −4417 −4682 436 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 405 −2170 −4497 −3510 −3299 −3769 −4347 −1223 −4924 375 −4729 −4142 −3306 3936 −2707 −2021 −3808 −3825 −4618 −3974 1346 437 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 406 761 −1334 68 978 −3637 −2817 809 −3388 1616 −3332 −2405 572 −751 1393 −940 880 −789 −1062 −3499 −2817 438 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 407 −2667 −4244 1961 −744 −4531 2730 −854 −4318 −915 −4245 −3370 1538 −3544 −1769 −1357 −2115 −493 −3853 −4418 −3658 439 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 408 1466 3113 −4382 −3829 −2304 −3540 −2788 −1834 −3490 −2196 1011 866 −3766 −3161 −3389 −329 935 1966 −2791 −2449 440 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 409 −1171 −3316 412 −140 −3637 −2817 976 −1458 861 −3332 −2405 473 −2910 1316 215 129 2374 −1381 −3499 −2816 441 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 410 −2304 −451 −4627 −4007 3602 −3848 −2618 −1533 −3605 432 672 −3475 −3883 −3202 −3396 −2010 −2243 370 −2405 2139 442 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 411 −3907 −3414 −6533 −6152 −3546 −6287 −5839 3217 −6046 595 −2331 −5939 −5988 −5757 −6057 −5610 −3881 2023 −5133 82 443 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 412 790 1969 −4777 −4348 −2968 2486 −3348 −785 −4017 −2826 −2194 448 −3951 −3670 −3899 −807 −2489 1776 −3442 −3115 444 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 413 −1507 −3497 −3894 −4200 −5869 −3595 −4454 −5770 −4397 −5909 −5041 −3759 −4351 4478 −4459 −959 −3263 −4597 −5954 −5675 445 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 414 −2565 −3197 −4765 −4794 −5469 −1282 −4553 −5224 −4762 −5456 −4558 −3914 3884 −280 −4789 395 6 −1383 −5678 −5465 446 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 415 −3978 −3624 −6358 −5756 −2482 −5738 −4608 878 −5427 2976 1708 −5437 −5425 −4697 −5115 −1639 −1600 −2294 −3876 −3823 447 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 416 −7875 −6321 −7259 −7641 −6426 −6309 −6538 −8612 −7781 −7895 −7930 −7574 −6736 −7627 −7209 −8364 −8007 −8416 6311 −6122 448 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 417 259 −3257 −5631 −5968 −5855 3636 −5191 −5659 −5782 −5315 −43 −4307 −4339 −5276 −5458 −2909 −3130 −4427 −6076 −6026 449 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 418 −188 −3252 −5668 −6022 −5929 −3522 −5224 −5788 −5864 −6041 −5081 −4312 −4334 −5314 −5517 3642 −3124 −4468 −6125 −6098 450 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 419 −3937 −3451 −6606 −6334 −4002 −6298 −6475 3965 −6299 −2737 −2721 −6104 −6130 −6206 −6432 −5734 −3949 30 −5766 −5281 451 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 420 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 452 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 421 −6234 −5148 −6596 −6958 1876 −6465 −2679 −5126 −6520 −4427 −4526 −5102 −6332 −5244 −5881 −5741 −6088 −5286 −1926 4688 453 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 422 10 −3182 −5823 −6163 −5898 −3463 −5215 −5716 −5868 −5982 −5009 −4285 −4279 −5307 −5491 1049 3703 −4395 −6119 −6099 454 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 423 −4994 −4428 −7368 −6797 772 −7026 −5656 −2020 −6572 3172 −1356 −6776 −6184 −5359 −6056 −6370 −1311 −1114 −4293 −4363 455 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 424 151 −3357 −5518 −5879 −6031 −1391 −5256 −5892 −5884 −6129 −5180 −4365 −4123 −5337 −5573 −3008 −3233 −4579 −6163 −6190 456 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 425 3585 −76 −5953 −6299 −5900 −3466 −5246 −5718 −5922 −5990 −5017 −4311 −4284 −5352 −5518 −628 −3056 −4396 −6122 −6109 457 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 426 2335 −2033 −4488 −3883 −2036 −3651 −2660 −1502 −3497 1390 315 −3362 −3761 −3130 −3320 −823 1243 395 −2553 −2213 458 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 427 −4457 −4009 −6802 −6193 2616 −6309 −4972 610 −5896 2458 209 −5975 −5766 −4965 −5498 −5521 −4332 −835 −3995 1086 459 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 428 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 460 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 429 2612 472 −5872 −6102 −5620 −2098 −5093 −5381 −5732 −5664 −4748 −4262 −4260 −5202 −5388 1752 1902 −1435 −5886 −5809 461 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 430 430 1087 −2441 −1346 −2572 −307 743 −2160 −1739 −212 −576 −2062 −3238 3683 −2108 −2124 −1911 −1989 −2884 −2418 462 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 431 −2120 −2921 −5674 −5112 −2717 −5049 −4050 1716 −4792 2048 1556 −4702 −4954 −4373 −4619 −4197 1898 503 −3707 −3428 463 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 432 3629 −3601 −5879 −6239 −6218 −877 −5483 −6134 −6147 −6344 −5431 −4660 −4649 −5621 −5787 −3290 −3510 −4842 −6211 −6375 464 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 433 1018 2365 1762 379 −3637 −2817 1089 −3387 −375 −1986 −2405 1644 −2910 897 −320 −544 −964 −2938 −3499 −2816 465 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 434 −1101 −3220 −1751 −262 −3498 −2850 −1511 −3217 898 −860 −505 −1503 3281 −361 −264 −264 −732 −1794 −3430 −2776 473 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 435 −1466 −3321 1471 1410 −3642 117 1359 −3392 317 −3337 −2410 1377 −2913 1172 −1568 961 −828 −2943 −3504 −1312 474 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 436 −4518 −5051 −5556 −3816 −6068 −4927 −2688 −5258 −6 −4837 −4203 −3600 −4829 150 4007 −1159 −4120 −5065 −4627 −4615 475 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 437 −4188 −4636 −5602 −4944 −6253 −4721 −4046 −6179 −2825 −5921 −5269 −4528 −5174 −3748 −4191 −1690 −4383 −5524 −5484 −5519 476 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 438 −1889 −2878 −1977 −1426 −3035 −1468 2360 −2680 −1325 −2211 −835 2293 −379 508 −1786 −149 200 1992 −3171 −2606 477 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 439 −3959 −3434 −6649 −6329 −3875 −6524 −6532 3066 −6309 487 −2567 −6181 −6194 −6170 −6448 −5919 −3945 2482 −5708 −5295 478 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 440 −4708 878 −7255 −6791 −2726 −6537 −5861 −1906 −6573 3235 −1509 −6587 −6085 −5474 −6118 −5929 −4674 −504 −4518 −4659 479 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 441 −3885 −3510 −6292 −5713 1957 −5727 −4605 2058 −5408 2414 −1461 −5391 −5428 −4743 −5135 −1616 −3803 −430 −3923 −3794 480 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 442 −3578 −3156 −6190 −5786 −3696 −5765 −5316 3605 −5620 −1128 −2525 −5463 −5654 −5443 −5637 −1663 359 1072 −4966 −4526 481 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 443 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 7152 −7196 −7139 −8047 −6473 −7685 482 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 444 −7014 −6306 4224 −5700 −7594 −5941 −6176 −8542 −6898 −7975 −7846 −5993 −6426 −6415 −6929 −7061 −7171 −8132 −6465 −7357 483 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 445 −6820 −6058 −7070 −7454 −7683 3860 −6792 −8642 −7678 −8092 −7936 −7259 −6560 −7508 −7152 −7196 −7139 −8047 −6473 −7685 484 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 446 2074 −3225 −5740 −6091 −5922 −3499 −5227 −5767 −5878 −6025 −5058 −4306 −4314 −5319 −5515 3168 −3097 −4443 −6127 −6105 485 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 447 2551 −4132 −6676 −6410 2806 −5642 −4373 −2425 −6103 1279 −1791 −5615 −5659 −5154 −5675 −4967 −4402 −3055 −3540 −2817 486 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 448 −5571 −5510 −5464 −5542 −4991 −5571 −5041 −5636 −4641 −1259 −4981 −5471 −5990 −4546 −4607 −5743 −5726 −5868 −5317 −4898 487 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 449 −5290 −4627 −7651 −7045 −154 −7513 −6025 −2069 −6860 3135 1997 −7253 −6355 −5467 −6255 −6940 −5102 −1478 −4412 −4668 488 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 450 −2617 −3226 −5575 −5905 −5785 −3498 −5134 −4289 −5670 −5916 −4997 −4263 −4308 −5215 −5366 −1426 4019 −4388 −6031 −5907 489 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 451 1563 −3339 −6536 −6241 −4109 −6241 −6449 2319 −6211 −2874 −2793 −6000 −6077 −6184 −6384 −5622 −3830 2812 −5869 −5323 490 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 452 −4063 −4579 −4421 −4655 −6179 −4548 −4778 −6468 −4363 −6356 −5760 −4489 −5188 4555 −4400 −4220 −1331 −5623 −5906 −5808 491 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 453 −1088 −5589 −752 3719 −6176 −1340 −3292 −6148 −3695 −6014 −5406 −2436 −4364 −2987 −4657 −899 −4024 −5506 −6215 −5141 492 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 454 −4707 −4179 −7117 −6536 −2603 −6772 −5546 1205 −6300 2876 1891 −6469 −6048 −5265 −2757 −6060 −4579 8 −4324 −4455 493 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −13 −9550 −6991 −894 −1115 −701 −1378 * * 455 −661 −3188 −5652 −6007 −5910 2743 −5192 −5742 −5850 −5999 −5024 −4256 −4276 −5274 −5494 2692 −3056 −4407 −6125 −6101 494 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 456 −1414 −2264 −3236 −2681 −2348 −3362 −2310 −1323 −2485 −1229 −1540 −1620 −3503 −630 −2695 2050 2945 −407 −2773 −2380 495 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 457 −491 −1961 −4486 −3851 656 −3694 −2565 1905 −3448 −481 4260 −3340 −3736 −3065 −3247 −1788 −2080 −565 561 −2081 496 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 458 −564 −1881 −4389 −3753 −411 −2111 −2470 2108 −3350 2217 542 −3242 −1247 −1296 −3154 −937 −1993 −557 −2338 −818 497 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 459 −4739 −5210 −4220 −4019 −6188 −5006 −3133 −5679 −1728 −5266 −4654 −3885 −5070 −663 −4163 −4635 −4460 −5436 −4956 −4939 498 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 460 −1859 −3332 2176 1662 −3653 −2826 547 −3404 −447 −1327 −2422 −281 −2922 2156 −260 −1738 −1799 −2954 −3515 1453 499 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 461 −1970 −3450 818 −118 −3771 2172 742 −3523 1425 −3463 −2543 1312 −3013 805 315 −1843 −1911 −3073 −3627 −2942 500 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 462 −4602 −4080 −7027 −6481 −2681 −6716 −5588 1460 −6221 2520 −1425 −6402 −6046 2476 −5865 −6018 −4493 −309 −4404 −4500 501 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 463 −697 −3338 −1723 −1171 −3663 −2843 823 −3409 2751 −3351 −2428 1887 −1087 −411 142 253 17 −2962 −3515 −2840 502 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 464 −508 −2733 −4958 −4645 −3515 −1510 −3753 −1905 −4311 −2151 −202 −3795 4000 −3989 −4205 −2780 −926 −2720 −3982 −3675 503 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 465 −1531 −3082 −6077 −5643 −3590 −5668 −5070 2883 −1664 −1114 −2455 −5319 −5545 −5258 −5444 −4906 −1178 2757 −4760 −4327 504 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 466 −3638 466 −6232 −5813 −3550 −5867 −5292 3665 −5646 −560 −2393 −5518 −5690 −5418 −5641 −5127 −3614 939 −4859 −38 505 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 467 −4630 −4091 −7087 −6550 3782 −6796 −5664 714 −6352 844 −505 −6485 −6090 −5370 −5975 −6109 −4520 1155 −4431 −4517 506 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 468 −4360 −3808 −6964 −6557 −3143 −6813 −6234 257 −6473 2551 −1884 −6497 −6226 −5790 −6315 −6208 −4305 2454 −4983 −4968 507 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 469 −4323 −3778 −6922 −6508 −3143 −6756 −6153 2761 −6415 1907 317 −6432 −6191 −5752 −6261 −6133 −4268 1694 −4958 −4931 508 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 470 −4779 −5848 −892 −3060 −6636 −4525 −4227 −7028 −4717 −6802 −6327 4376 −5123 −4007 −5533 −4529 −4963 −6384 −6313 −5849 509 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 471 −6510 −5994 −6070 −6452 −7251 −5898 −6379 −8368 −7089 −7883 −7678 4452 −6410 −6832 −6854 −6751 −6807 −7779 −6351 −7068 510 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 472 −1538 −4183 3319 1047 −4471 −300 −95 −4258 −1891 −4186 −3306 1073 −3499 320 −2468 −1381 −2579 −3792 −4359 −2367 511 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 473 −6803 −6045 −7057 −7441 −7671 3860 −6780 −8628 −7666 −8080 −7923 −7245 −6548 −7495 −7140 −7178 −7123 −8032 −6462 −7673 512 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 474 −7100 −5902 −6970 −7339 −3194 −6279 −4485 −6826 −7282 −6139 −6213 −6465 −6584 −6588 −6717 −7063 −7139 −6872 −3783 4945 513 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −619 −1178 * * 475 −1314 −3313 −5039 −5322 −5885 −3551 −4925 −5645 −2746 −5913 −5008 −4155 −4343 −4901 −4922 −2940 4007 −4448 −6032 −5944 514 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 476 −3893 −3371 −6597 −6296 −4087 −6475 −6627 2196 −6289 −2227 −2759 −6133 −6192 −6276 −6491 −5875 −3887 3409 −5929 −5382 515 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 477 −4917 −6083 −2614 3919 −6686 −4565 −4135 −6991 −4414 −6721 −6267 −3384 −5137 −2025 −5067 −4603 −5042 −6448 −6286 −5827 516 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 478 −4984 −5192 −6260 −5405 −6240 −5261 −4357 −6356 −3138 −6048 −5561 −5048 −5615 −4100 4225 −5112 −5068 −2975 −5549 −5604 517 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 479 2638 325 −1215 −1193 −305 −3455 −2272 −1557 −1045 682 894 −2786 −3515 −1596 −2771 −1339 −1966 −1055 −2474 991 518 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 480 −4030 −3609 −6609 −6400 −3937 −6043 −6251 4011 −6320 −2712 −2740 −6063 −6045 −6132 −6351 −5549 −4077 −788 −5562 −5130 519 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 481 −3237 −3518 −4079 −3274 −3507 −4212 4961 −3235 −1613 256 −58 −901 −4264 −2337 −66 −2444 −3109 −3106 −3619 −3221 520 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 482 −5124 −5281 −5605 −5973 −7088 3838 −6016 −7671 −6503 −7457 −6958 −5744 −5910 −2344 −6374 −5346 −5523 −6733 −6350 −6953 521 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 483 930 −3124 −1807 2107 −3364 −2877 −1545 −3058 −1162 −3105 2169 −1551 1921 639 −535 −1390 −1807 −1736 1084 −2731 522 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 484 −490 −3316 1098 1747 −3638 −2817 482 −2797 108 −3332 −2406 2169 −2910 1035 −573 −1393 888 −2938 −3500 −867 523 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 485 814 −3363 −1751 1620 −3690 −2707 −1542 −3431 −1106 −3381 −117 −1522 −2979 3123 291 543 −1857 −2991 −3551 −2881 524 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 486 −264 −3093 −1818 370 −3322 −2879 −1549 −3010 −479 −1596 −711 −1559 2041 −1127 2224 906 −836 −488 −3334 −124 525 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 487 -6291 −5271 −6494 −6839 −1480 −6354 −1157 −5379 −6475 −4685 −4776 −5263 −6345 −5399 −5923 −5880 −6194 −5515 −2191 4901 526 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −13 −9550 −6991 −894 −1115 −701 −1378 * * 488 −6510 −5994 −6070 −6452 −7251 −5898 −6379 −8368 −7089 −7883 −7678 4452 −6410 −6832 −6854 −6751 −6807 −7779 −6351 −7068 527 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 489 −4119 −6225 3992 −75 −6330 −3804 98 −6355 −3752 −6170 −5646 −363 −4410 −2978 −4819 −3703 −4230 −5780 −6375 −5193 528 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 490 −3861 −3366 −6568 −6280 −4066 −6328 −6548 3902 −6257 −2798 −2748 −6068 −6125 −6224 −6436 −5726 −3204 −654 −5883 −5351 529 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −842 −1178 * * 491 1953 −3302 −855 −1136 −1694 −890 743 −3368 −452 −3316 −2391 −120 828 2272 −362 −971 53 −1028 −3487 −2806 530 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −619 −1519 * * 492 694 −3246 −1727 −596 −3535 −1348 −512 −3009 −450 −828 3372 863 516 386 499 −487 −732 −260 −3448 −2785 531 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 493 −4610 1105 −6521 −6808 −3706 −5094 −4730 −6455 −6383 −6131 −5857 −2463 −5708 −6082 −6034 −4831 −5011 −5865 6217 −3278 532 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 494 −2192 −3688 2691 −902 −4006 −3040 733 −3762 118 −3696 −2788 2708 −3191 284 392 −1003 −2138 −3311 −3858 −3163 533 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 495 −6213 −5135 −6592 −6945 −1195 −6465 −2671 −5078 −6487 −1797 −4476 −5093 −6325 −5228 −5857 −5729 −6066 −5250 −5262 3836 534 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9550 −10592 −894 −1115 −701 −1378 * * 496 52 −3327 −1710 −1161 −3649 −2834 738 −3397 1072 −3442 −2417 59 −1838 1192 −670 9 2832 −2950 −3509 −2830 535 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 497 1182 −3308 −75 934 −3628 −516 −756 −3379 1440 −528 −2397 −654 −2902 2112 18 −1027 −1774 −869 −3491 −2808 536 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 498 −4366 −3863 −6861 −6350 −2813 −6532 −5557 −1257 −6153 2852 799 −6213 −5980 −5347 −5868 −5821 −1332 675 −4490 −4513 537 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 499 534 −2066 −4488 −3891 −850 −3675 −2705 −573 −3515 −1134 −1321 −3385 3680 −3158 −3354 −1576 −1835 −160 −2611 −2268 538 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 500 1136 −3308 −100 534 −3629 −1385 −1337 −3380 1198 −3324 −468 586 −1686 2253 307 −502 −809 −2930 −3491 −2808 539 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 501 2420 −2147 −4409 −3851 −592 −3575 −2776 −1426 −3506 −2137 −1491 −477 −3777 −3170 −3389 −1641 850 2260 −2753 −2409 540 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 502 −5318 −4643 −7588 −7040 3618 −7449 −5566 −2117 −6841 2217 417 −7066 −6348 −5450 −6233 −6867 −5131 −3019 −4158 −4002 541 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 503 283 678 316 −1176 −3551 2609 −54 −3283 −596 −3263 −557 1036 −2926 −1048 −1593 437 −1286 −2707 −3458 −2793 542 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9539 −10581 −894 −1115 −701 −1378 * * 504 1172 −2879 −1255 −1404 −3039 1552 −1623 −1912 43 27 −742 −785 −122 −1246 −5 −660 537 714 −3169 −2601 543 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −155 −9539 −3310 −894 −1115 −701 −1378 * * 505 168 −3192 1294 1378 −3513 246 18 −3264 1005 −3208 −2281 1105 −1492 323 −304 −1599 1121 −2814 −3375 −2692 544 — −148 −501 237 45 −381 398 105 −627 210 −466 −720 275 394 45 96 358 116 −370 −295 −250 — −534 −1695 −10429 −234 −2740 −412 −2008 * * 506 120 1125 1556 1257 −1015 −295 1718 −1288 536 −3305 −2379 254 −844 32 −590 −353 −522 −659 −3473 −2790 547 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −131 −9517 −3548 −894 −1115 −983 −1018 * * 507 −710 −177 −1009 928 −3512 −559 −1352 −3263 974 −2470 −2281 1286 −1120 2059 1077 −1116 696 −1220 675 −2692 548 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −38 −9389 −5358 −894 −1115 −1962 −428 * * 508 1420 202 −4147 −3517 −972 −1350 104 33 −3134 −1588 735 −3049 945 −2775 −2968 1280 −1838 1825 −2195 −404 549 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −40 −9355 −5266 −894 −1115 −363 −2168 * * 509 −1059 −3007 −638 973 220 −2838 −1510 −1499 429 1366 −906 −1525 −2928 1754 167 −1209 261 −62 −3256 −2644 550 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9484 −10526 −894 −1115 −1314 −742 * * 510 −126 3208 −4392 −3767 −1888 −3624 −2524 198 −3376 −1364 −274 −3270 −3682 −3010 −3195 1352 2356 769 −2402 −2058 551 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9484 −10526 −894 −1115 −719 −1349 * * 511 −1329 2072 −4340 −1658 1935 −3577 2398 −61 −843 471 −330 −3210 −3627 −2940 −143 −2660 −1974 −81 3627 824 552 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9517 −10559 −894 −1115 −983 −1018 * * 512 −626 −2032 1111 −591 −3587 −2796 −1455 −1237 1667 −2216 −417 −372 −2889 33 2532 −784 −1104 −59 −3462 −2783 553 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9517 −10559 −894 −1115 −983 −1018 * * 513 1143 −337 −6156 −5870 −3921 −3813 −5371 107 −5688 −2944 −2745 −5225 −5343 −5472 −5657 −4401 −3466 3457 −5166 −4738 554 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 514 −356 −86 −1662 1664 −914 −2788 −386 −3356 563 −3301 −2375 805 −2882 367 552 1144 1516 −2907 −3469 −2787 555 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 515 −1883 118 −506 922 −3675 −2831 107 −3426 −1101 −3372 −2449 850 −2938 1289 −1613 389 2831 −2978 −3542 −2855 556 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 516 925 −3078 −1785 1662 −3315 −842 −1521 −3007 −1144 −3060 −2195 −1527 325 −1096 −692 734 1006 856 −3320 793 557 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 517 450 −3284 −438 811 −3604 1308 30 146 529 −3299 −2373 530 −2882 693 −1536 −242 646 189 −3468 −2786 558 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 518 −131 −5188 958 2748 −5424 −912 −2788 −5291 −2848 −5182 −4416 −2172 −4026 2916 −3585 −1969 −3446 −4785 −5368 −4453 559 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 519 −5219 −4566 −7550 −6984 639 −7379 −5749 −2032 −6792 3204 −1321 −7075 −6305 −5431 −6204 −6797 −5043 −708 −4265 −4283 560 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 520 1203 −3289 1142 1536 −3610 −2790 −1448 −3361 1367 −3305 −343 −499 −2883 1503 811 −1696 −1755 −1255 −3472 −2790 561 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 521 904 −3287 1101 2037 −3608 −2788 −1447 −3359 429 −1036 −2376 335 −2881 1398 −677 −94 −980 −1589 −3470 −2788 562 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 522 2435 −2606 −5242 −4770 −2960 −4168 −3719 −818 −4434 −1753 −2071 −4137 −4424 −4098 −4287 −2159 664 2558 −3631 −3278 563 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 523 −5255 −4571 −7584 −6979 1880 −7427 −5937 −2032 −6788 2790 2658 −7159 −6301 −5415 −6193 −6839 −5040 −1168 −4358 −4589 564 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 524 1079 −3287 989 1856 −3608 −1579 −508 −1678 470 −1582 −1586 150 −2881 187 −284 −740 1058 −2774 −3470 −2787 565 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −983 −1018 * * 525 534 −3287 −144 1270 −3608 −2788 293 −3359 999 −1440 −2376 512 −2881 1717 1260 −504 −638 −37 −3470 −2788 566 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −51 −9513 −4893 −894 −1115 −983 −1018 * * 526 1975 −2759 −5585 −5075 −3040 −4974 −4154 2312 −4790 765 −2038 −4654 −4945 −4498 −4688 −1747 −3056 1608 −3927 −3550 567 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9464 −10506 −894 −1115 −1444 −661 * * 527 1225 −3250 209 1056 −3571 −892 −663 −1231 306 −3266 −2339 1413 −1081 1147 −549 564 319 −2761 −3433 −2750 568 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −519 −9464 −1734 −894 −1115 −1444 −661 * * 528 1881 −2851 −318 543 −216 −1147 −1035 −1070 687 −1957 −1942 −813 −2468 1096 399 70 −381 −768 −3039 −2362 569 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −4 −8950 −9992 −894 −1115 −161 −3246 * * 529 514 −3287 1018 −98 −3608 −974 2548 −3359 −425 −2291 −2376 1916 −2881 1082 522 −862 234 −2909 −3470 −237 570 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −162 −9513 −3253 −894 −1115 −983 −1018 * * 530 −44 1529 −1543 −420 −3474 −1398 1178 −2010 1267 −3171 −894 −93 1812 1470 −3 134 −87 −1693 −3342 −793 571 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9354 −10396 −894 −1115 −225 −2791 * * 531 −790 −3381 2768 1433 −3700 −2112 −1526 −3453 −351 −3396 −2472 693 −1942 1798 −639 −776 −1842 −3002 −3563 −2876 572 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9535 −10577 −894 −1115 −701 −1378 * * 532 −1843 393 −1728 −1178 −3506 −284 1690 −3230 612 −3222 −746 −122 −2923 2326 2483 −1741 −503 −1064 −163 −941 573 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9535 −10577 −894 −1115 −701 −1378 * * 533 −1221 −2434 −4973 −4343 −1243 −1226 −3082 −533 −3953 2627 3217 −3864 −1616 −3509 −3736 −3310 −2572 −1707 −2834 −2554 574 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9535 −10577 −894 −1115 −701 −1378 * * 534 1450 −1930 −4104 −3491 −1896 −3556 2495 247 −3149 −1787 −1138 −3113 −3617 −2827 −3053 1185 679 1734 −2391 −189 575 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9535 −10577 −894 −1115 −701 −1378 * * 535 −4417 −3969 −6340 −5980 3761 −5864 −3411 −2532 −5614 1659 101 −5162 −5604 −4752 −5220 −5039 −4311 −774 953 65 576 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 536 −2071 −3283 −6466 −6137 −3983 −6276 −6219 3157 −6088 −646 −2689 −5934 −6035 −6036 −6240 −5635 −1188 2500 −5658 −5137 577 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 537 −4698 −6039 −2358 3889 −6557 −4372 −3888 −6779 −4115 −6525 −6046 −3128 −4945 −770 −4766 −4363 −4812 −6239 −6218 −5631 578 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 538 −307 −3323 −6487 −6148 −3826 −6243 −6121 −154 −6081 112 −2550 −5932 −6005 −5943 −6184 −5595 −3805 3550 −5492 −5057 579 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 539 −2025 293 −4164 −3541 −1842 −3549 1126 700 1261 −753 2388 −1249 −3601 −2842 −1477 −2625 −412 2460 −2339 −1992 580 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 540 −1563 −3152 −5842 −5253 −2458 −5138 −4100 −1563 −4909 2261 3761 −4842 −5002 −4347 −4664 −4291 3 589 −3613 −3441 581 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 541 520 −3326 1125 81 −3645 −63 −133 −3396 −362 −2267 −2416 −1447 2861 −424 −1577 322 −1792 −2947 −3509 −2825 582 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 542 −130 −2971 −1951 −1392 −3156 −2947 −1611 −2811 2278 −2922 1553 −1661 369 1369 1618 −1876 −1842 194 636 −521 583 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 543 74 −3147 1222 881 −117 −579 934 −3118 −466 −379 3446 −452 −2917 −1053 −1594 −945 −1766 −2737 3368 17 584 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 544 −4169 −6191 4038 285 −6370 −2745 −3346 −6411 −3828 −6222 −5705 −2453 −4459 −3051 −4886 −3763 −4286 −5829 −6365 −5255 585 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 545 1832 −1886 −4143 −3522 −1844 −1044 −2404 1737 −853 91 950 −3115 −3598 598 −3045 −1244 −143 564 −2340 401 586 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 546 −7151 −6137 −7088 −7470 −7647 −6121 −6786 −8695 −7673 −8087 −7998 −7367 4335 −7540 −7140 −7568 −7423 −8217 −6429 −7632 587 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 547 476 −3285 36 1975 −3605 −2735 791 −3355 1122 −1411 −2374 −1424 1747 −464 −790 −850 −1348 −282 −3469 −2787 588 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 548 −1976 −171 −3149 −2571 −706 −1143 −2137 −1639 827 2330 −1286 −2547 −1485 −598 −1066 390 −1226 283 −2522 −2133 589 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 549 −4076 −3935 −6596 −6196 −2660 −5489 −5140 −2143 −5807 3215 1093 −5660 −5498 −5016 −5450 −1793 −1843 −2804 −4252 −4308 590 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 550 1078 −3286 369 −606 −3606 −924 386 −346 1531 −1218 −2375 −784 −1792 470 1582 287 −1045 −1330 −3469 −2787 591 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 551 870 −3288 955 762 −3609 −2788 −1447 −3359 1869 −3304 −2377 575 −2881 916 −134 −154 649 −927 −3471 −2788 592 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 552 −2451 −2244 −4811 −850 1617 −4050 −2936 2017 −3799 1315 1090 −3697 −2122 −3398 −3598 −3147 −2391 1908 −2742 −2425 593 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 553 1760 1317 −4381 −3879 −2618 1475 −2979 −2170 −3574 −503 −1846 −3366 −3799 −3267 −2945 1023 1661 −342 −3089 −2748 594 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 554 469 −203 −307 −441 −3608 −778 −624 −3359 2219 −1751 −2376 −27 169 1274 1396 −405 −1753 −2909 −3470 −2788 595 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9513 −10555 −894 −1115 −701 −1378 * * 555 2140 531 −2674 −2109 −2303 −1382 −215 −974 −1956 −84 −1461 −2220 −573 48 1298 782 −1879 −10 −2680 −2251 596 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9540 −10522 −894 −1115 −701 −1378 * * 556 −925 1036 −4355 −3719 2743 −3561 −2433 844 −809 1845 −352 −3206 −3611 −2939 −3117 −2497 35 −641 −2298 −1957 597 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9480 −10522 −894 −1115 −701 −1378 * * 557 1679 −3271 −1635 1223 −3592 210 597 −3343 −28 −3287 −2361 890 −582 1048 −1519 1028 −1737 −2893 −3455 −2772 598 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9456 −10498 −894 −1115 −701 −1378 * * 558 1025 −3243 803 28 −3564 −1358 −1403 −1721 374 −3259 −1543 1050 402 1597 −146 737 186 −1876 −3426 −2744 599 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9456 −10498 −894 −1115 −701 −1378 * * 559 −327 −1900 −605 −1094 1259 −2758 −1418 −3245 74 −3213 −2299 −1401 −1804 2740 2365 −287 −1717 −2817 −3399 −801 600 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9443 −10486 −894 −1115 −701 −1378 * * 560 −2174 −5296 1116 −1204 −5520 −1310 −2820 −5404 45 −5286 −4551 3932 −4039 −2467 −3701 −3182 −1194 −4890 −5474 −4526 601 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −3 −9403 −10445 −894 −1115 −701 −1378 * * 561 212 −859 −622 −707 493 653 −1369 −3227 1180 −3187 −2268 762 −2803 −913 308 1455 −263 −1536 −3366 947 602 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −5 −8809 −9851 −894 −1115 −701 −1378 * * 562 1180 −2467 −1321 −427 −2668 2005 −1030 −1181 303 −2431 −1589 −1053 −1043 −623 100 231 −1262 −487 −2727 840 603 — * * * * * * * * * * * * * * * * * * * * — * * * * * * * * 0

MEGA 

What is claimed is:
 1. A method of converting α-ketoisovalerate to isobutyraldehyde comprising a. providing a polypeptide wherein said polypeptide comprises: (i) at least 80% identity to SEQ ID NO: 53, 56, 58, 59, or 63 or an active fragment thereof; and (ii) said polypeptide has α-ketoisovalerate decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than 1, and thiamine diphosphate cofactor activation constant (K_(c)) of about 20 μM or less; and b. contacting said polypeptide with α-ketoisovalerate under conditions wherein isobutyraldehyde is produced.
 2. The method of claim 1 wherein the contacting occurs within a recombinant host cell and wherein the polypeptide is heterologous to recombinant host cell.
 3. The method of claim 2 wherein the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Issatchenkia, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces.
 4. The method of claim 2 wherein the recombinant host cell is Saccharomyces cerevisiae.
 5. The method of any one of claims 2-4 wherein the recombinant host cell further comprises heterologous polynucleotides encoding polypeptides which catalyze the substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; and (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate.
 6. The method of claim 5 wherein the host cell further comprises a heterologous polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol.
 7. The method of claim 5, wherein the recombinant host cell further comprises reduced or eliminated pyruvate decarboxylase activity.
 8. A method of producing isobutanol comprising: a. providing a recombinant host cell comprising an isobutanol production pathway, the production pathway comprising a polypeptide wherein said polypeptide comprises: (i) at least 80% identity to SEQ ID NO: 53, 56, 58, 59, or 63 or an active fragment thereof; and (ii) said polypeptide has α-ketoisovalerate decarboxylase activity, a specificity ratio for α-ketoisovalerate to pyruvate greater than 1, and thiamine diphosphate cofactor activation constant (K_(c)) of about 20 μM or less; and b. contacting the recombinant host cell with a carbon substrate under conditions whereby isobutanol is produced.
 9. The method of claim 8 wherein the recombinant host cell is Saccharomyces cerevisiae. 